Steps.csv

"CSHP-2008-Estecio-pdb.prot4974.txt","2","<R>LowC dNTP mix (10X)"," -PRh(appendices)"
"CSHP-2008-Estecio-pdb.prot4974.txt","2","<R>MCA reaction buffer (10X)"," -PRh(appendices)"
"CSHP-2008-Estecio-pdb.prot4974.txt","2","<R>MCAM wash solution 1"," -PRh(appendices)"
"CSHP-2008-Estecio-pdb.prot4974.txt","2","<R>SSC (20X) is diluted 100-fold in H2O to prepare this wash solution, which is 0.2X SSC."," -PRh(appendices)"
"CSHP-2008-Estecio-pdb.prot4974.txt","2","<R>Prehybridization solution for MCAM"," -PRh(appendices)"
"CSHP-2008-Estecio-pdb.prot4974.txt","2","<R>React2 buffer (10X)"," -PRh(appendices)"
"CSHP-2008-Estecio-pdb.prot4974.txt","2","<R>TE buffer (pH 8.0)"," -PRh(appendices)"
"CSHP-2008-Estecio-pdb.prot4974.txt","3","This method was adapted from an original protocol by Toyota et al. (1999). An online version is also available at http://www.mdanderson.org/departments/methylation/."," :X1ir(x)"
"CSHP-2008-Estecio-pdb.prot4974.txt","3","An online version is also available at http://www.mdanderson.org/departments/methylation/."," r(x)"
"CSHP-2008-Estecio-pdb.prot4974.txt","3","1. Digest 5 μg of genomic DNA using 100 units of SmaI for 16 h at 25°C."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","3","2. Add 20 units of XmaI and incubate the reaction for 6 h at 37°C."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","3","3. Bring the reaction volume to 500 μL with H2O and add one volume of 1.5:1 phenol:chloroform (pH 9.0). Vortex the mixture for 1 min, centrifuge for 10 min at 13,000 rpm, and transfer the supernatant to a 1.7-mL microcentrifuge tube."," :AP2;AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","3","add one volume of 1.5:1 phenol:chloroform (pH 9.0). Vortex the mixture for 1 min, centrifuge for 10 min at 13,000 rpm, and transfer the supernatant to a 1.7-mL microcentrifuge tube."," AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","3","Bring the reaction volume to 500 μL with H2O"," AP2"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","4. Precipitate the DNA by adding 1/10th volume of 3 M sodium acetate and two volumes of 100% ethanol."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","5. Incubate the sample at −70°C for 1 h, and centrifuge for 30 min at 13,000 rpm. Pour out the ethanol and air dry the DNA pellet."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","6. Resuspend the DNA in 20 μL of TE and determine the DNA concentration using a spectropho- tometer."," :AP1;AQ1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","determine the DNA concentration"," AQ1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","i. Dilute the RXMA (or RMCA) primers to 100 μM."," :AP2"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","ii. Combine 50 μL of RXMA24 with 50 μL of RXMA12 (or RMCA24 and RMCA12)."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","8. Incubate the adaptor mix for 3 min at 65°C, and cool it to room temperature over 60 min. This mixture can be stored for up to 6 mo at -20°C."," :AP1ew"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","cool it to room temperature"," e"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","This mixture can be stored for up to 6 mo at -20°C."," w"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","i. Combine the following reagents: 500 ng 10 μL 3 μL 400 units to 30 μL Digested DNA (Step 6) Adaptor mixture (Step 8) 10X ligase buffer T4 DNA ligase H2O"," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","ii. Incubate the ligation reaction for 16 h at 16°C."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","10. Prepare the MCA reactions using the following reagents: 10 μL 100 pmol 15 units 1.2 μL 5 μL 3 μL to 97 μL 10X MCA reaction buffer RXMA24 (or RMCA24) primer Taq DNA polymerase dNTP mix (25 mM) DMSO (RMCA primers only) Ligation mixture (Step 9.ii) H2O DMSO is not needed with RXMA primers. Typically, four MCA reactions per tested sample are necessary to generate enough PCR product for labeling."," :AP1ma,AP3"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","DMSO is not needed with RXMA primers."," AP3"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","Typically, four MCA reactions per tested sample are necessary to generate enough PCR product for labeling."," ma"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","11. Incubate the reaction for 5 min at 72°C, and perform PCR as follows: Number of cycles 20 Temperature Time 95°C 72°C (for RXMA) or 77°C (for RMCA) 1 min 3 min 1"," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","12. Analyze 10 μL of the PCR products on a 1.5% agarose gel to check the quality of the amplification. A relatively strong smear, ranging from 300 bp to 2 kb, should be visible for RXMA primers. Expect a smear rang- ing from 200 bp to 1 kb when using RMCA primers."," :AQ2;AI3ev"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","Analyze 10 μL of the PCR products on a 1.5% agarose gel"," AQ3"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","check the quality of the amplification"," AI3"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","A relatively strong smear, ranging from 300 bp to 2 kb, should be visible for RXMA primers. Expect a smear rang- ing from 200 bp to 1 kb when using RMCA primers."," e"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","This hybridization protocol is optimized for the Human CpG-island 12K Array (HCGI12K) from University Health Network (www.microarrays.ca)."," :X3x"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","13. Combine PCR products of replicate samples and purify them using the QIAquick PCR Purification kit according to the manufacturer’s recommendation. Elute the samples in 30 μL of 1X TE buffer."," :S2r(x);AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","Combine PCR products of replicate samples and purify them using the QIAquick PCR Purification kit according to the manufacturer’s recommendation."," -MBdh(QIAquick PCR Purification Kit Instructions)"
"CSHP-2008-Estecio-pdb.prot4974.txt","4","14. Quantify the DNA yield using a spectrophotometer."," :AQ2"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","i. Assemble each labeling reaction as follows (35-μL total volume): 25 μL (5 μg) MCA product (Step 11) in H2O 10 μL (7.5 μg/μL) Random octomer primer (8N)"," :AP1mr(11)"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","ii. Denature the nucleic acids by incubating at 100°C for 5 min, and then immediately trans- fer the tubes to ice."," :AP1m"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","iii. Add the following reagents to the nucleic acids (to a 50-μL total volume): 5 μL 5 μL 3 μL 2 μL 10X React2 buffer 10X LowC dNTP mix Cy3-dCTP (or Cy5-dCTP) Klenow fragment (stock 40 units/μL)"," :AP1ma"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","Typically, Cy5-dCTP mix is used for tumor DNA and Cy3-dCTP is used for normal (reference) DNA."," a"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","iv. Incubate the reactions for 2 h at 37°C."," :AP1m"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","16. Increase the reaction volume to 100 μL with H2O, and purify each sample using a PCR purification kit (QIAquick) as follows:"," :AP2"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","i. Add 500 μL of PBI buffer to the column in the PCR purification kit, let it stand for 5 min, centrifuge the column for 1 min at 13,000 rpm, and discard the flow-through."," :AP1m"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","ii. Wash the column with 700 μL of PE buffer, centrifuge for 1 min at 13,000 rpm, discard the flow-through, and centrifuge once again at 13,000 rpm for 1 min."," :AP1m"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","iii. Place the column in a clean 1.7-mL microcentrifuge tube, and elute twice with 50 μL of H2O, waiting 2 min between centrifugations."," :AP1m"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","17. Read the samples in a spectrophotometer at the following wavelengths (use the full volume of labeled sample): Cy3= 550 nm; Cy5= 650 nm."," :AQ1x"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","use the full volume of labeled sample"," x"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","18. Calculate the concentration of incorporated dyes as follows: Cy3 = A550/0.15 = pmol/μL of Cy3 dye incorporated Cy5 = A650/0.25 = pmol/μL of Cy5 dye incorporated Optimal amounts for hybridization are between 250 pmol (minimum) and 400 pmol (maximum) of incor- porated dyes."," :AI1e"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","Optimal amounts for hybridization are between 250 pmol (minimum) and 400 pmol (maximum) of incor- porated dyes."," e"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","19. Prehybridize the slides for 45 min at 42°C in prehybridization solution for MCAM."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","20. Combine equal picomole amounts of labeled tumor and normal amplicons and bring to 500 μL with H2O. Reduce the volume to a 15-μL concentrate using a centrifugal filter unit (Microcon YM-30) as follows:"," :AP2"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","i. Centrifuge the column for 12 min at 14,000g."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","ii. Invert the column in a new microcentrifuge tube and centrifuge it for 3 min at 5000 rpm to collect the sample."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","iii. Transfer the sample to a new microcentrifuge tube and bring the volume to 15 μL with H2O, if necessary. This step typically takes 12 min at 14,000g."," :AP2e"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","This step typically takes 12 min at 14,000g."," e"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","21. Heat a large beaker of H2O to boiling on a hot plate."," :AP2"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","22. Rinse microarray slides in room temperature H2O."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","23. Place the slides in boiling H2O for 1 min and in 4°C ethanol for another 1 min."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","24. Immediately centrifuge the microarrays for 6 min at 600 rpm in a benchtop centrifuge."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","5","25. Add 10 μL of human Cot1 DNA (total 10 μg) and 25 μL of 2X SDS hybridization solution to the sample from Step 20. Mix gently."," :AP1r(20)"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","26. Denature the DNA (from Step 25) for 2 min at 95°C, and hold the tubes in a 60°C water bath."," :AP1r(25)"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","27. Place the microarray slides in a hybridization chamber. Add 50 μL of MCAM wash solution 2 under- neath each side of the slide."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","28. Transfer the labeled DNA onto the array area and place a coverslip over the array."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","29. Close the hybridization chamber tightly and incubate it in the dark for 16 h in a 60°C water bath. The hybridization chamber should be submerged in water, but direct contact with the bottom of the water bath should be avoided."," :AP1x"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","The hybridization chamber should be submerged in water, but direct contact with the bottom of the water bath should be avoided."," x"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","30. Warm two 500-mL bottles of MCAM wash solution 1 and 1 500-mL bottle of MCAM wash solu- tion 2 to 60°C."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","31. Place the slides, with coverslips facing down, in a slide-staining dish containing MCAM wash solu- tion 1 at 60°C. Keep the slides at a 45° angle to the bottom of the dish to allow the coverslips to detach from the microarrays."," :AP1x"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","Keep the slides at a 45° angle to the bottom of the dish"," x"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","32. Place a slide rack in a slide-staining dish filled with prewarmed MCAM wash solution 1. Transfer the slides into the dish and place it on a rocker platform set to 100 rpm for 10 min."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","33. Transfer the slides to a new dish filled with prewarmed MCAM wash solution 2 and place it on a rocker platform set to 100 rpm for 10 min."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","34. Rinse the slides by submerging the slide rack 10-20 times in H2O."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","35. Immediately centrifuge the slides for 6 min at 600 rpm in a benchtop centrifuge."," :AP1"
"CSHP-2008-Estecio-pdb.prot4974.txt","6","36. Scan the slides."," :AP1,AV1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","1. On the day of the experiment, obtain a leukapheresis pack from a blood bank. Dilute 109 leuko- cytes with 1X PBS (1:1). Pipette 15 mL of LSM into a 50-mL conical tube. Carefully layer the lym- phocyte suspension over the LSM."," :AP1x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","A clear boundary between the two phases is critical for the removal of erythrocyte contamination."," x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","2. Centrifuge at 2000 rpm for 25 min at 20°C. After centrifugation, the order of layers from top to bottom is plasma/platelets, lymphocytes, Ficoll-Hypaque, and granulocytes/erythrocytes."," :AP1i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","the order of layers from top to bottom is plasma/platelets, lymphocytes, Ficoll-Hypaque, and granulocytes/erythrocytes."," i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","3. Aspirate off the top layer and leave the lymphocyte interphase intact. Transfer the cells in the inter- phase to a 250-mL conical tube. Wash with 1X PBS and centrifuge at 800 rpm for 12 min at 20° C-23°C to remove the residual platelets. Remove supernatant, resuspend cells in 1X PBS, and repeat the wash once to remove remaining platelets."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","4. Resuspend in 20-30 mL of 1X PBS and then count the cells. Aliquot 0.5 × 106 cells for fluorescence- activated cell sorting (FACS) analysis."," :AP1w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","20-30 mL"," w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","Use the CD4+ T Cell Isolation Kit II (human) (Miltenyi Biotec). Alternatively, Dynabeads Untouched Human CD4 T Cells Kit can be used for Steps 5-21."," :AP1r(5-21)r(x)w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","Alternatively, Dynabeads Untouched Human CD4 T Cells Kit can be used"," w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","for Steps 5-21."," r(5-21)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","Keep the cells cold."," :AP1x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","5. Aliquot 108 cells from Step 4."," :AP1er(4)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","from Step 4."," r(4)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","The final yield of purified CD4+ T cells is ~20%."," e"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","6. Centrifuge the cell suspension at 300g for 10 min."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","7. Resuspend the cell pellets in 40 μL of T cell isolation buffer per 107 cells."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","8. Add 10 μL of biotin-antibody cocktail (from T cell isolation kit) per 107 cells."," :AP1i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","The biotin-antibody cocktail contains monoclonal antibodies against CD14, CD16, CD19, CD36, CD56, CD123, and glycophorin A."," i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","9. Mix well and incubate for 10 min at 4°C-8°C or on ice."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","10. Add 30 μL of T cell isolation buffer per 107 cells."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","11. Add 20 μL of anti-biotin microbeads (from T cell isolation kit) per 107 cells."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","12. Mix well and incubate for 15 min at 4°C-8°C."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","13. Wash cells with T cell isolation buffer by adding 10X-20X the final labeling volume. Centrifuge at 300g for 10 min. Pipette off supernatant completely."," :AP1w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","14. Resuspend the cells completely into appropriate volume of T cell isolation buffer. Use 3 mL for 250-300 million cells."," :AP1x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","completely"," x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","3","15. Proceed to magnetic separation using an LS column (Step 16)."," :CB(16)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","16. Place LS column in the magnetic field of a suitable MACS separator."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","17. Prepare the column by rinsing with 3 mL of T cell isolation buffer. The buffer will run through the column quickly."," :AP1i, CB(12)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","This step can be performed during antibody incubation (Step 12)."," CB(12)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","18. Apply cell suspension onto the column: load ~250-300 million cells per column. Allow the cells to pass through and collect effluent. This fraction contains unlabeled cells and represents the enriched CD4+ T cell fraction."," :AP1i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","This fraction contains unlabeled cells and represents the enriched CD4+ T cell fraction."," i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","19. Wash the column three times with 3 mL of T cell isolation buffer. For each wash, add buffer once the column reservoir is empty. Collect the entire effluent in the same tube (from Step 18)."," :AP1ar(18)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","For each wash, add buffer once the column reservoir is empty."," a"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","Collect the entire effluent in the same tube (from Step 18)."," r(18)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","20. Count cells and check CD4+ T cell purity. Recovery is typically ~20%."," :AQ1, AI1ei"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","Recovery is typically ~20%."," e"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","Purified CD4+ T cells with ~ 95%-98% purity are used for native chromatin preparation."," i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","21. Centrifuge at 300g for 10 min."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","This step is only for analyzing histone modifications. To analyze target sites of proteins that may not be as stably or directly bound to chromatin as histones, the chromatin should be cross-linked using formaldehyde and sonicated to 200- to 300-bp length."," :X1i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","22. Resuspend 20-22 × 106 purified human CD4+ T cells from Step 21 in 1 mL MNase digestion buffer kept at room temperature and add protease inhibitor cocktail to 1X final concentration."," :AP1r(21)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","from Step 21"," r(21)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","23. Add 0.3 U of MNase and incubate for 5 min at 37°C."," :AP1, AI4"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","Determine the amount of MNase to use for each cell type experimentally."," AI4"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","24. Stop the reaction by adding MNase stop buffer to a final concentration of 10 mM Tris (pH 7.6), 10 mM EDTA."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","25. Sonicate three times for 20 sec each in ice water."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","26. Dialyze against 400 mL of RIPA buffer for 2 h at 4°C."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","27. Centrifuge for 10 min at 4°C. Transfer supernatant to a new tube, add glycerol to a final concen- tration of 5%, and store at −80°C."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","28. To check the size of the isolated chromatin, transfer 20 μL of supernatant to a new tube. Add 4 μg of RNase A and incubate for 5 min at room temperature. Add 3 μL of 10% SDS and 5 μL of 20 mg/mL proteinase K. Incubate at 65°C for 2 h and purify the DNA by phenol:chloroform extraction and ethanol precipitation. Analyze on a 2% agarose gel."," :AP1a"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","The above digestion procedure will primarily yield mononucleosomes. To obtain other fragment lengths, vary the MNase amounts and digestion time."," a"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","29. Continue to the ChIP experiment (Step 30)."," :CB(30)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","Do NOT block beads with any nonspecific DNA or RNA."," :X1x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","30. Place 40 μL of Dynabeads Protein A into each of two microcentrifuge tubes. Wash each with 600 μL of 1X PBS. Place the tubes in a magnetic stand and aspirate off the PBS. Add 100 μL of 1X PBS to each tube. Add 4 μg of antibody to one tube and 4 μg of preimmune serum to the other. Rotate for 4-6 h at 4°C."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","31. Using the magnetic stand, aspirate the supernatants and wash the beads in each tube twice with 0.2 mL of 1X PBS to remove free IgGs (5 min each wash)."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","32. Remove the supernatant from the beads. Add 500 μL of chromatin extract (from 107 cells) to the beads in each tube. Rotate at 4°C overnight."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","4","33. Wash the beads (10 min each wash):"," :AP1w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","i. Twice with 1 mL of RIPA buffer"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","ii. Twice with 1 mL of RIPA buffer + 0.3 M NaCl"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","iii. Twice with 1 mL of LiCl buffer"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","iv. Once with 1 mL of 1X TE + 0.2% Triton X-100"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","v. Oncewith1mLof1XTE"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","For less stringent washing, reduce the concentration of NaCl added to the RIPA buffer and/or perform only a single wash at each step (Steps 33.i-33.v)."," w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","34. Resuspendthebeadsin100μLof1XTEpertube.Add3μLof10%SDSand5μLof20mg/mL proteinase K to each tube. Incubate overnight at 65°C."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","i. Vortex briefly and transfer the supernatants to new tubes using a magnetic stand."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","ii. Wash the beads with 100 μL of 1X TE and 0.5 M NaCl. Combine each wash solution with the corresponding supernatant."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","iii. Extract each tube once with 200 μL of phenol:chloroform."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","iv. Add 2 μL of 20 mg/mL glycogen, 20 μL of 3 M NaOAc (pH 5.3), and 500 μL of ethanol to each tube to precipitate the DNA."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","36. Wash each pellet once with 70% ethanol."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","37. Resuspend each pellet in 50 μL of 1X TE. Use 2 μL of diluted (1:5) DNA for real-time PCR confirmation."," :AP1x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","Use 2 μL of diluted (1:5) DNA for real-time PCR confirmation."," x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","38. Before moving to the next step, perform real-time PCR to ensure that ChIP has worked well."," :AQ1e"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","Enrichment of the region of interest should be significantly higher in the ChIP sample obtained using antibodies than in the sample prepared using preimmune serum."," e"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","39. Repair DNA ends to generate blunt-ended DNA using the DNA End-Repair Kit (Epicentre). Combine:"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","5","40. Use MinElute PCR Purification Kit or phenol:chloroform extraction to purify DNA. Elute or resus- pend DNA in 30 μL of 1X TE, pH 7.4."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","41. Add “A” to 3′ ends."," :AP1w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","i. Combine:"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","ii. Purify using MinElute PCR Purification Kit or phenol:chloroform extraction."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","iii. Elute with or resuspend in 10 μL of 1X TE, pH 7.4."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","Alternatively, Klenow fragment exo- can be used for adding “A” bases."," w"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","42. Perform linker ligation. Combine:"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","43. Purify DNA using MinElute PCR Purification Kit. Elute with 20-25 μL of buffer EB."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","44. Perform size selection:"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","i. Load 20 μL of linker-ligated DNA onto a 2% E-Gel and perform electrophoresis."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","ii. Cut the gel around the 200-400 bp region."," :AP1ix"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","DNA is not visible on the gel at this step"," i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","Load only one sample per gel to avoid contamination."," x"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","iii. Extract the DNA using MinElute Gel Extraction Kit. Elute with ~11.5 μL of buffer EB."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","45. Amplify the DNA using Illumina PCR primers PE 1.0 and PE 2.0 and 2X Phusion HF Master Mix."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","i. Combine:"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","ii. Denature at 98°C for 30 sec, followed by 18 amplification cycles (98°C, 10 sec; 65°C, 30 sec; 72°C, 30 sec.)"," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","iii. Check 2.5 μL of product on a 2% agarose gel. If the band is not clearly visible, perform three more cycles and check again."," :AP1v"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","If the band is not clearly visible"," v"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","i. Load the amplified DNA on a 2% agarose gel and perform electrophoresis."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","ii. Excise the band at the 200-400 bp region."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","iii. Purify the DNA using a MinElute Gel Extraction Kit and elute in 20 μL of buffer EB."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","6","iii. Purify the DNA using a MinElute Gel Extraction Kit and elute in 20 μL of buffer EB."," :AP1"
"CSHP-2009-Cuddapah-prd.prot5237.txt","7","iv. Measure the DNA concentration using a spectrometer."," :AQ1ir(x)"
"CSHP-2009-Cuddapah-prd.prot5237.txt","7","The DNA can be used for cluster generation using Illumina flow cells and sequencing-by-synthesis on an Illumina Genome Analyzer,"," i"
"CSHP-2009-Cuddapah-prd.prot5237.txt","7","following manufacturer’s instructions."," r(x)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","1. Grow 2 × 107 cells of the cell type of interest in an appropriate medium and under suitable con- ditions until they are 70%-80% confluent."," :S2e"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","2. Add 0.86 mL of 37% formaldehyde (to a final concentration of 2%) directly to a cell culture dish containing 15 mL of medium. Swirl gently to mix. Incubate for 10 min at room temperature."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","3. Add 1.057 mL of 2 M glycine (to a final concentration of 0.125 M) to the dishes. Swirl gently to mix. Incubate for 5 min at room temperature."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","4. Aspirate the medium. Wash the cells with 5 mL of ice-cold PBS containing protease inhibitor cocktail."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","5. Prepare 0.2X trypsin diluted in PBS, and add 5 mL of it to the cells. Incubate in 5% CO2 for 5 min at 37°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","6. Neutralize the trypsin with 10 mL of 10% fetal bovine serum."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","7. Scrape the cells from the plate. Transfer them to 50-mL tubes."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","Optionally, scrape a second time with PBS containing protease inhibitor cocktail."," :AP1o"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","8. Collect the cells by centrifuging at 1300 rpm for 8 min at 4°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","9. Wash twice with 10 mL of PBS containing protease inhibitor cocktail. Working on ice, pipette gen- tly to make a single-cell suspension."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","After resuspending the second time, count the cells before centrifugation."," :AQ1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","4","10. Add 10 mL of cellular lysis solution containing protease inhibitor cocktail to the cells. Mix gently by pipetting. Incubate for 10 min on ice."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","Monitor lysis by observing an aliquot under a microscope. These conditions should be sufficient for most cell types. However, certain cell types might need different incubation times and/or different strengths of the buffer for complete lysis."," :W1a"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","11. Centrifuge at 1800 rpm for 5 min at 4°C. Discard the supernatant."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","At this point, the cell pellet can be snap-frozen in liquid nitrogen and stored at -80°C until use."," :AP1o"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","12. Resuspend the pellet in 500 μL of 1.14X restriction enzyme buffer (appropriate for the restriction enzyme to be used in Step 16)."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","13. Pellet the cells by centrifugation. Resuspend in 500 μL of 1.14X restriction enzyme buffer. Transfer to 1.5-mL tubes."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","14. Add 7.5 μL of 20% SDS (to a final concentration of 0.3% SDS). Mix by gentle pipetting. Shake gently for 1 h at 37°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","15. Add 50 μL of 20% Triton X-100 (to a final concentration of 1.8%). Shake gently for 1 h at 37°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","16. Add 1200 units of restriction enzyme (i.e., 12 μL of 100 units/μL). Incubate with gentle shaking overnight at 37°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","17. Add 40 μL of 20% SDS (to a final concentration of 1.6%). Incubate with shaking for 30 min at 65°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","18. Transfer the sample to a 15-mL tube containing 7 mL of 1.15X ligation buffer."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","19. Add 400 μL of 20% Triton X-100 (to a final concentration of 1%). Incubate with occasional gen- tle shaking for 1 h at 37°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","20. Add 50 μL of T4 DNA ligase. Incubate for 4 h at 16°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","21. Incubate for 30 min at room temperature."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","22. Remove 10 μL from the sample (from Step 21). Store at -20°C."," :AP1ir(21, 37)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","from Step 21"," r(21)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","This sample will serve as the “input” sample for subsequent analysis"," i"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","(see Step 37)"," r(37)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","23. Remove an 800-μL aliquot from the sample (from Step 21). Add it to 7.2 mL of ChIP diluent (i.e., a 10-fold dilution)."," :AP1ir(21)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","from Step 21"," r(21)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","Multiple ChIP reactions can be performed from one 3C ligation reaction. The same ligation reaction can be split and used for ChIP analyses with different antibodies."," i"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","24. Add 4-10 μg of the antibody of choice per immunoprecipitation. Rotate end-over-end overnight at 4°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","i. Mix Protein G-conjugated beads:Protein A-conjugated beads (1:3) in a 1.5-mL tube."," :AP1i"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","Each immunoprecipitation experiment will need 100 μL of this bead mix. The choice of beads will strictly depend on the type of antibody being used."," i"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","ii. Add 1 mL of 0.5% BSA in PBS and mix."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","iii. Place the tube in a magnetic stand for 1 min."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","iv. Remove the solution by vacuum suction."," :AP1x"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","Be careful not to aspirate the beads."," x"
"CSHP-2009-Tiwari-pdb.prot5168.txt","5","v. Remove the tube from the stand. Add 1 mL of 0.5% BSA in PBS. Invert the tube to resus- pend beads."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","vii. Resuspend the beads in 1 mL of 0.5% BSA in PBS. Rotate end-over-end overnight at 4°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","26. Add 100 μL of the blocked magnetic bead solution to each immunoprecipitate sample (from Step 24). Incubate with rotation for 3 h at 4°C."," :AP1r(24)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","from Step 24"," r(24)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","27. Prechill one 1.5-mL tube for each immunoprecipitate."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","28. Transfer ~1.5 mL of each immunoprecipitate to a separate prechilled tube. Place the tubes in the magnetic stand to collect the beads. Remove the supernatant. Add another aliquot of the remain- ing immunoprecipitate. Repeat until all the beads have been collected."," :AP1mw"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","Repeat until all the beads have been collected."," m"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","Alternatively, pellet the magnetic beads in a centrifuge at 2000 rpm for 5 min. Aspirate some of the super- natant, leaving ~1 mL in the tube. Resuspend the pellet with the remaining supernatant and transfer to a chilled tube. Let the tube sit in the magnetic stand for 1 min to collect the beads. Aspirate the remaining supernatant."," w"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","29. Remove the tubes from the magnetic stand. Place on ice."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","30. Wash the beads four times with 500 μL of low-salt immune complex wash buffer containing pro- tease inhibitor cocktail. Agitate gently to resuspend the beads. Place the tubes in the magnetic stand to collect the beads. Remove the supernatant."," :AP1amx"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","Wash the beads four times"," m"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","Use fresh buffer for each wash."," x"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","If the beads are stuck on the bottom of the tube, place on the magnetic stand momentarily to move the beads to the side and continue to invert."," a"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","31. Wash the beads once with 500 μL of high-salt immune complex wash buffer containing protease inhibitor cocktail. Agitate gently to resuspend the beads. Place the tubes in the magnetic stand to collect the beads. Remove the supernatant."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","32. Wash the beads once with 1 mL of TE buffer containing protease inhibitor cocktail. Collect on the magnetic stand. Remove the supernatant."," :AP1a"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","The bead pellet is relatively loose in the TE buffer. Remove the supernatant by pipetting manually instead of vacuum aspiration."," a"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","33. Centrifuge at 960g for 3 min at 4°C. Remove any residual TE buffer."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","34. Add 210 μL of bead eluting buffer. Incubate for 15 min at 65°C. Vortex briefly every 2 min."," :AP1w"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","Alternatively, shake gently for 15 min in a shaking incubator pre-set to 65°C."," w"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","35. Centrifuge at 16,000g for 1 min at room temperature."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","36. Transfer 200 μL of the supernatant to a new 1.5-mL tube."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","37. Thaw the inputs (from Step 22). Add 190 μL of bead eluting buffer."," :AP1r(22)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","from Step 22"," r(22)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","38. Reverse-cross-link samples (from Step 36) and inputs (from Step 37) overnight at 65°C."," :AP1ar(36, 37)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","from Step 36"," r(36)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","from Step 37"," r(37)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","Cover the tubes in the heat block with aluminum foil to reduce evaporation."," a"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","39. Dilute the SDS by adding 200 μL of TE buffer. Add 8 μL of 10 mg/mL RNase A. Incubate for 2 h at 37°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","40. Add 8 μL of 10 mg/mL proteinase K. Incubate for 2 h at 50°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","41. Add an equal volume of phenol:chloroform:isoamyl alcohol to the samples. Shake well. Centrifuge at 10,000 rpm for 15 min. Transfer the upper phase to a fresh tube. Repeat this step with the upper phase."," :AP1x, CL(41)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","equal volume of phenol:chloroform:isoamyl alcohol"," x"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","Repeat this step with the upper phase."," CL(41)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","42. Precipitate the DNA by adding 0.1 volume of 3 M sodium acetate, 1 μL glycogen, and 2 volumes of 100% ethanol."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","43. Wash with 0.4 volume of 70% ethanol. Vortex gently after adding the ethanol."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","44. Dissolve the pellet in 15 μL of nuclease-free H2O."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","6","45. Test the success of the ChIP reaction using 1 μL of undiluted immunoprecipitate by PCR with primers specific for a chromatin region known to be occupied by the protein of interest in the cell type under investigation."," :AQ3"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","46. Using standard cloning protocols, clone the 3C-ChIP products (from Step 44) into a vector that has enzyme overhangs similar to those generated in the 3C assay."," :AP1ir(44)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","from Step 44"," r(44)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","This step typically involves cloning an “IgG/no antibody 3C-ChIP product” and “Plus antibody 3C-ChIP prod- uct,” in addition to regular cloning controls."," i"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","47. Use the ligated vector to transform high-efficiency competent bacterial cells."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","48. For experimental samples, spread the surface of LB-agar plates containing the antibiotic of choice (i.e., LB-ampicillin or LB-kanamycin agar plates), with 45 μL of X-gal and 9 μL of IPTG per 100-mm dish before plating the transformed cells."," :AP1o"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","49. The next day, count the number of blue and white colonies in the bacterial plates from samples immunoprecipitated with the specific antibody as well as the controls (i.e., no antibody or IgG)."," :AQ1ix"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","White colonies represent bacteria-harboring plasmids with inserts, whereas blue colonies represent bacteria with plasmids without the insert."," i"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","In an ideal 6C experiment, the number of white colonies should be several- fold higher in samples immunoprecipitated with the specific antibody than in controls."," x"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","This indicates that the pull-down with a specific antibody enriched specific protein-occupied genomic regions."," i"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","50. Pick several white colonies (i.e., containing the insert) from the bacterial plates."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","51. Use each such colony to inoculate LB liquid medium containing the appropriate antibiotic. Incubate in a shaking incubator at 220 rpm overnight at 37°C."," :AP1"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","52. Purify the plasmids using the PureLink HQ 96 Mini Plasmid DNA Purification Kit according to the manufacturer’s instructions."," :AP1r(x)w"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","Alternatively, standard plasmid preparation protocols or kits can also be used."," w"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","53. Screen the purified plasmids (from Step 52) by digestion with the same restriction enzyme used for Step 16, using standard restriction digestion protocols."," :AP1r(52, 16)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","from Step 52"," r(52)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","for Step 16"," r(16)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","54. Separate the resulting DNA fragments by electrophoresis in a 1% agarose gel containing SYBR Safe. Use an appropriately sized DNA ladder for reference. Visualize using UV light."," :AP1; AV1cr(Troubleshooting)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","See Troubleshooting."," cr(Troubleshooting)"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","55. Sequence the plasmids showing more than one insert."," :AP1x"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","Ideally, multiple inserts mean interacting partners."," x"
"CSHP-2009-Tiwari-pdb.prot5168.txt","7","56. Sequence the inserts using primers from two different ends of the vector (e.g., T3 and T7 promoter-specific primers for pBluescript II RI Predigested Vector)."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","2","The RQPS probe is linear DNA consisting of two physically linked DNA sequences, one of which corresponds to part of the query gene and the other to the reference gene, each with a SNV to differentiate the probe from the correspon- ding part of the genome (see Fig. 2)."," :X1i"
"CSHP-2010-Liu-pdb.prot5491.txt","3","1. Select a gene/genomic locus with a known CN in the genome as the reference gene."," :DS3a"
"CSHP-2010-Liu-pdb.prot5491.txt","3","Although any gene/genomic locus can be used as a reference, it is best to use genes unlikely to have CNV or SNPs among individuals, such as the exons of the genes that will give phenotypes in case of amplification or loss of heterozygosity. We routinely use mouse and human Notch2 as reference genes in our studies. SRY is another (haploid) possibility in males."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","3","i. To avoid the interference of naturally occurring SNPs and DNA variations, select coding DNA sequence and avoid the third codon position."," :X1x"
"CSHP-2010-Liu-pdb.prot5491.txt","3","avoid the third codon position."," x"
"CSHP-2010-Liu-pdb.prot5491.txt","3","ii. Consult a SNP database to confirm the sequence if such a database is available."," :AV1i"
"CSHP-2010-Liu-pdb.prot5491.txt","3","The SNP information among different mouse strains can be found in the mouse Phenome database at Jax (http://phenome.jax.org). Links to various other SNP databases can be found at the website for the Center for Human and Clinical Genetics, Leiden University Medical Center (http://www.humgen.nl/ SNP_databases.html)."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","3","iii. Perform a BLAST search with the Basic Local Alignment Search Tool (BLAST) (http:// blast.ncbi.nlm.nih.gov/Blast.cgi) to help ensure that the selected sequences are unique in the genome under study."," :AV1"
"CSHP-2010-Liu-pdb.prot5491.txt","3","i. Design three primers: two PCR amplification primers (F1 and R1) and one sequencing primer (S1)."," :AV2"
"CSHP-2010-Liu-pdb.prot5491.txt","3","ii. Choose primers with a high score (>85) in the Assay Design Software."," :DS2"
"CSHP-2010-Liu-pdb.prot5491.txt","3","iii. Addacommontail(5-AGCGCTGCTCCGGTTCATAGATT-3)tothe5endofthePCRampli- fication primer that is in the opposite direction of the sequencing primer (F1 in Fig. 2)."," :AV1r(32-40)x"
"CSHP-2010-Liu-pdb.prot5491.txt","4","Do not add this tail to the primer that goes the same direction as the sequencing primer."," x"
"CSHP-2010-Liu-pdb.prot5491.txt","4","see Steps 32-40"," r(32-40)"
"CSHP-2010-Liu-pdb.prot5491.txt","4","4. Repeat Steps 2 and 3 for the query gene (to prepare F2, R2, S2)."," :CL(2-3)3i"
"CSHP-2010-Liu-pdb.prot5491.txt","4","The SNP and primer design for the reference and query genes are independent from each other, because the pyrosequencing reactions will be performed in different wells."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","4","5. Selectastufferfragmentthatis~200-500bpinlengthanddesigntwoDNAprimers(Rf-1andQr-1):"," :AP1iw"
"CSHP-2010-Liu-pdb.prot5491.txt","4","~200-500bpinlength"," w"
"CSHP-2010-Liu-pdb.prot5491.txt","5","i. The stuffer DNA fragment can be any DNA that has no significant homology to either the query or reference sequence."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","5","ii. Each primer should be ~70 bp long, with 20 bp on the 3 end complementary to the stuffer DNA sequence and 50 bp on the 5 end matching the reference (for Rf-1) and query (for Qr-1) DNA fragments (see Fig. 2A)."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","5","iii. Incorporate the SNVs chosen in Step 2 into these primers."," :AP1r(2)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","chosen in Step 2"," r(2)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","6. Use the primers designed in Step 5 to prepare the following amplification mix:"," :AP1r(5)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","designed in Step 5"," r(5)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","7. Perform the following PCR cycling program:"," :AP1a"
"CSHP-2010-Liu-pdb.prot5491.txt","5","To conserve energy, the samples can also be held at 10oC overnight (instead of 4oC)."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","5","8. AnalyzeanaliquotofthecompletedreactionbyDNAgelelectrophoresistoconfirmthatthePCR worked."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","5","9. (Optional) If the designed reference and query fragments are >50 bp, design another pair of primers (Rf-2 and Qr-2 in Fig. 2) with 3 ends that overlap Rf-1 and Qr-1, respectively. Repeat the PCR, using 1 μL of product from the reaction in Step 7 as template."," :AV1o; CL(7)ir(7)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","(Optional)"," o"
"CSHP-2010-Liu-pdb.prot5491.txt","5","Repeat the PCR"," CL(7)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","from the reaction in Step 7 as template"," r(7)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","The RQPS probe can be prepared with other DNA cloning methods or direct DNA synthesis, but the strategy described here (sequential PCR) is simple and economical."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","5","10. (Optional) Analyze an aliquot of the completed reaction by DNA gel electrophoresis to confirm that the PCR worked."," :AP1o"
"CSHP-2010-Liu-pdb.prot5491.txt","5","(Optional)"," o"
"CSHP-2010-Liu-pdb.prot5491.txt","5","accord- ing to the manufacturer’s instructions."," r(x)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","11. PurifythefinalPCRproductwithaPCRpurificationkit(e.g.,QIAquickPCRPurificationKit)accord- ing to the manufacturer’s instructions."," :AP1ar(x)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","If nonspecific bands appear in the gel, then the desired band should be cut from the gel and purified with a gel extraction kit."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","5","(Optional)"," o"
"CSHP-2010-Liu-pdb.prot5491.txt","5","i. Mix the following components:"," :AP1o"
"CSHP-2010-Liu-pdb.prot5491.txt","5","ii. Incubate the mixture for 15-30 min at 70°C. 7 μL 1 μL 1 μL 1 μL"," :AP1iow"
"CSHP-2010-Liu-pdb.prot5491.txt","5","for 15-30 min"," w"
"CSHP-2010-Liu-pdb.prot5491.txt","5","High-fidelity Taq DNA polymerase will not produce a 3 overhang A in the PCR product."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","5","13. Clone the purified PCR product into a TA vector according to the manufacturer’s instructions."," :AP1r(x)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","according to the manufacturer’s instructions"," r(x)"
"CSHP-2010-Liu-pdb.prot5491.txt","5","14. Screen for positive clones."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","5","15. Purify and sequence the plasmid to confirm that the sequence is correct."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","5","16. Linearize ~5 μg of plasmid with a restriction enzyme that will not cut inside either the query or reference DNA fragment."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","17. Gel-purify the linearized plasmid with a gel extraction kit."," :AP1ai"
"CSHP-2010-Liu-pdb.prot5491.txt","6","This purified, linearized plasmid will be used as the RQPS probe."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","6","Purified RQPS probes can be stored at −20°C until use."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","6","We routinely use the following method described by Laird et al. (1991)."," r(x)"
"CSHP-2010-Liu-pdb.prot5491.txt","6","Various protocols or commercial kits can be used to purify genomic DNA from tissues and cells. We routinely use the following method described by Laird et al. (1991)."," :X1r(x)"
"CSHP-2010-Liu-pdb.prot5491.txt","6","18. Digestthetissuesorcellsin500μLoftissuelysisbufferfor2horovernightwithagitationat55°C."," :AP1ac"
"CSHP-2010-Liu-pdb.prot5491.txt","6","500 μL of tissue lysis buffer should be sufficient to fully digest a 2- to 3-cm-long mouse tail. Bigger tissues may need more tissue lysis buffer; adjust the volume accordingly."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","6","If digestion is incomplete after an overnight incubation, add more proteinase K and agitate for ~2 h until the tissue is fully digested."," c"
"CSHP-2010-Liu-pdb.prot5491.txt","6","19. Centrifugethedigeststopelletthetissuedebris.Transferthesupernatanttoanewmicrocentrifuge tube."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","20. Add an equal volume of 100% isopropanol and mix well to precipitate genomic DNA."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","21. Centrifuge the tubes at 13,000g for 2 min in a microcentrifuge to pellet the genomic DNA"," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","22. Discard the supernatant and add 750 μL of 70% ethanol."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","23. Centrifuge the tubes at 13,000g for 1 min and discard the supernatant."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","24. Centrifuge the tubes briefly to collect the remaining 70% ethanol to the bottom of the tube. Carefully remove the ethanol with a pipette."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","25. Air-dry the pellet for ~2 min."," :AP1x"
"CSHP-2010-Liu-pdb.prot5491.txt","6","Do not over-dry the genomic DNA, or it will be extremely difficult to resuspend."," x"
"CSHP-2010-Liu-pdb.prot5491.txt","6","i. Add 200-500 μL of TE buffer for RQPS to the DNA pellet."," :AP1w"
"CSHP-2010-Liu-pdb.prot5491.txt","6","200-500 μL of TE buffer"," w"
"CSHP-2010-Liu-pdb.prot5491.txt","6","ii. Vortex."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","iii. Incubate the DNA/TE mixture for ~2 h at 55oC."," :AP1a"
"CSHP-2010-Liu-pdb.prot5491.txt","6","The amount of TE to use depends on the amount of genomic DNA. Because the reference and query fragments that will be amplified for pyrosequencing are very short, shearing of genomic DNA by vortexing is not a concern and is probably beneficial. Purified genomic DNA can be stored short-term at 4°C and long-term at −20°C."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","6","27. Measure the concentration of genomic DNA and RQPS probe with a spectrophotometer."," :AP1ir(28)"
"CSHP-2010-Liu-pdb.prot5491.txt","6","It is NOT necessary to know the exact concentration of either the genomic DNA or RQPS probe"," i"
"CSHP-2010-Liu-pdb.prot5491.txt","6","see Step 28"," r(28)"
"CSHP-2010-Liu-pdb.prot5491.txt","6","i. For Mixture I, prepare a 1:1 molar ratio of RQPS probe to genomic DNA."," :AP1a"
"CSHP-2010-Liu-pdb.prot5491.txt","6","For a RQPS probe that is 3-4 kb long, 0.3-0.4 pg of probe gives a 1:1 ratio when mixed with 0.3 μg of mouse or human genomic DNA."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","6","ii. For Mixture II, prepare a 2:1 molar ratio of RQPS probe to genomic DNA."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","6","iii. Bring the final volume of each mixture to 10 μL with H2O."," :AP2a"
"CSHP-2010-Liu-pdb.prot5491.txt","6","Theoretically, one mixture should be enough to derive the copy number of the query sequence. However, to assess the quality of data with an R2 value (see Step 52), it is highly recommended to use at least two different mixtures. It is NOT necessary to achieve an exact 1:1 or 2:1 ratio in the mixture, because this ratio will be measured by the pyrosequencing reactions in the following steps."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","6","29. Thoroughly mix the RQPS probe/genomic DNA by vortexing, and centrifuge briefly to bring all samples to the bottom of the tubes."," :AP1aix"
"CSHP-2010-Liu-pdb.prot5491.txt","6","it is extremely important to mix the RQPS probe and genomic DNA thoroughly!"," x"
"CSHP-2010-Liu-pdb.prot5491.txt","6","Pyrosequencing measures relative ratios"," i"
"CSHP-2010-Liu-pdb.prot5491.txt","6","The mixtures of RQPS probe and genomic DNA can be stored short-term at 4°C or indefinitely at −20°C."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","7","This can be achieved easily by labeling one strand of PCR product with biotin"," a"
"CSHP-2010-Liu-pdb.prot5491.txt","7","A pyrosequencing reaction needs to be done on single-stranded DNA template. This can be achieved easily by labeling one strand of PCR product with biotin (Steps 30 and 31), followed by streptavidin-mediated, single-strand purifica- tion (Steps 32-40). To reduce the cost, a three-primer PCR system is employed, in which one primer is tailed with a common motif that can be bound by a UBP. (Fig. 2; Fakhrai-Rad et al. 2002)."," :X1ar(30-31)r(32-40)r(x)x"
"CSHP-2010-Liu-pdb.prot5491.txt","7","needs to be done on single-stranded DNA template."," x"
"CSHP-2010-Liu-pdb.prot5491.txt","7","Steps 30 and 31"," r(30-31)"
"CSHP-2010-Liu-pdb.prot5491.txt","7","Steps 32-40"," r(32-40)"
"CSHP-2010-Liu-pdb.prot5491.txt","7","Fakhrai-Rad et al. 2002"," r(x)"
"CSHP-2010-Liu-pdb.prot5491.txt","7","For each mixture in Step 29"," r(29)"
"CSHP-2010-Liu-pdb.prot5491.txt","7","i. Combine the following components for Reaction I (amplifying the fragment containing the reference SNV):"," :AP1r(29)"
"CSHP-2010-Liu-pdb.prot5491.txt","7","ii. Combine the following components for Reaction II (amplifying the fragment containing the query SNV):"," :AP1r(29)x"
"CSHP-2010-Liu-pdb.prot5491.txt","7","If two mixtures are made for each sample, four PCRs will be required (two for each mixture). If multiple samples need to be processed, prepare a master mix for each reaction and perform the amplification in a 96-well plate."," x"
"CSHP-2010-Liu-pdb.prot5491.txt","7","31. Perform the following PCR cycling program:"," :AP1a"
"CSHP-2010-Liu-pdb.prot5491.txt","7","To conserve energy, the samples can be held at 10oC overnight (instead of 4oC)."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","7","We have successfully used the same PCR cycle parameters for many different SNVs. Therefore, the optimization of PCR conditions may not be necessary for most SNVs. The PCR product can be stored short-term at 4°C or indefinitely at −20°C."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","7","Purify the Single-Stranded PCR Product with Streptavidin-Coated Sepharose Beads (~15 min for a 96-well plate)"," a"
"CSHP-2010-Liu-pdb.prot5491.txt","7","The simplest method to purify the single-strand PCR product from a large number of reactions (in a 96-well plate form) is to use the PyroMark Q96 Vacuum Prep Workstation."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","7","32. Fill three troughs of the Vacuum Prep Worktable with 70% ethanol, denaturation solution, and washing buffer, respectively."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","7","PCR product from Step 31"," r(31)"
"CSHP-2010-Liu-pdb.prot5491.txt","7","i. Add the following components to each well with PCR product:"," :AP1r(31)"
"CSHP-2010-Liu-pdb.prot5491.txt","7","ii. Mix well."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","7","iii. Incubate with constant shaking for 10 min at room temperature."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","34. During the incubation in Step 33, add the following to each well of a PyroMark Q96 Plate: 10 μL of 5 μM sequencing primer (S1 or S2 in Fig. 2) and 40 μL of RQPS annealing buffer."," :AP1ir(33), CB(33)"
"CSHP-2010-Liu-pdb.prot5491.txt","8","During the incubation in Step 33"," CB(33)"
"CSHP-2010-Liu-pdb.prot5491.txt","8","This is not the 96-well plate"," i"
"CSHP-2010-Liu-pdb.prot5491.txt","8","from Step 33."," r(33)"
"CSHP-2010-Liu-pdb.prot5491.txt","8","35. Continuing from Step 33, turn on the vacuum and use the hand-held Vacuum Prep Tool (VPT) to pick up the Sepharose beads."," :AP1r(33)"
"CSHP-2010-Liu-pdb.prot5491.txt","8","Continuing from Step 33"," r(33)"
"CSHP-2010-Liu-pdb.prot5491.txt","8","36. Move the VPT to the trough with 70% ethanol and wash the beads for 5 sec."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","37. Move the VPT to the trough with denaturation solution for 5 sec."," :AP1i"
"CSHP-2010-Liu-pdb.prot5491.txt","8","The PCR product is denatured and the nonbiotinylated strand is removed in this step."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","8","38. Move the VPT to the trough with washing buffer for 5 sec."," :AP1i"
"CSHP-2010-Liu-pdb.prot5491.txt","8","This step neutralizes the sample."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","8","39. MovetheVPTtothePyroMarkQ96PlatepreparedinStep34andshutoffthevacuumjustbefore the filter tips touch the surface of the liquid."," :AP1r(34)x"
"CSHP-2010-Liu-pdb.prot5491.txt","8","It is important to shut off the vacuum JUST before the tips touch the surface of the liquid in the PyroMark Q96 Plate. If it is shut off too early, the beads may be lost or fall to other wells; if it is not shut off when it touches the liquid, some or all of the liquid will be aspirated."," x"
"CSHP-2010-Liu-pdb.prot5491.txt","8","40. Gently shake the VPT in the buffer to release the beads."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","41. Denature the biotinylated, single-stranded DNA by heating the plate in a thermocycler for 2 min at 80°C-90°C."," :AP1i"
"CSHP-2010-Liu-pdb.prot5491.txt","8","This step disrupts possible secondary structure in the DNA."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","8","42. Cool the samples to room temperature to allow the annealing of sequencing primers to the single-stranded DNA."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","43. Set up the plate layout in the Assay Design Software by typing in sample and SNV information."," :AV1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","44. FillthePSQ96MAcassettewiththeamountofPyroMarkGoldQ96Reagentrecommendedbythe Assay Design Software."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","45. PlacethePyroMarkQ96Plateintothepyrosequencingmachineinthecorrectorientationandstart the pyrosequencing reactions."," :AP1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","46. Collectreadoutsfromthepyrogramsforthefollowingparametersforeachsample,andenterthe values into an Excel file: m1 (the ratio of the probe reference SNV in Mixture I), m2 (the ratio of the probe reference SNV in Mixture II), n1 (the ratio of the probe query SNV in Mixture I), and n2 (the ratio of the probe query SNV in Mixture II)."," :AV1ir(t)"
"CSHP-2010-Liu-pdb.prot5491.txt","8","Examples of pyrograms and readouts are shown in Figure 2D."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","8","See Troubleshooting."," r(t)"
"CSHP-2010-Liu-pdb.prot5491.txt","8","47. Calculate the following parameters (i.e., the ratios of genomic DNA SNVs) for each sample: (1−m1), (1−m2), (1−n1), (1−n2)."," :AV1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","48. Calculate the following parameters for each sample: m1/(1−m1), m2/(1−m2), n1/(1−n1), n2/(1−n2)."," :AV1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","49. MakeanXYscatterplotinExcelwithonedatapointat(n1/[1−n1],m1/[1−m1])andtheseconddata point at (n2/[1−n2], m2/[1−m2])."," :AV1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","50. Add the trend line (linear, y = kx; k is the slope of the line) and set the intercept to zero. Display the linear equation and the R2 value."," :AV1"
"CSHP-2010-Liu-pdb.prot5491.txt","8","51. To calculate the CN of the query gene, multiply k by the CN of the reference gene."," :AV1a"
"CSHP-2010-Liu-pdb.prot5491.txt","8","For example, if the copy number of the reference gene is two, double the value of k to get the copy number of the query gene."," a"
"CSHP-2010-Liu-pdb.prot5491.txt","9","52. Check the value of R2 to assess the quality of the data."," :AQ2ir(t)"
"CSHP-2010-Liu-pdb.prot5491.txt","9","The closer R2 is to 1, the better the data."," i"
"CSHP-2010-Liu-pdb.prot5491.txt","9","See Troubleshooting."," r(t)"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","at least two independent biological isolates per strain."," x"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","1. Streak the yeast strains of interest onto YPD agar plates. Use at least two independent biological isolates per strain."," :AP1aix"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","It is impor- tant to have well-separated single colonies."," x"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","Colonies should be visible in 2-3 d if the strains have no growth defects and are grown on rich media."," a"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","Strains with variable-sized colonies should be carefully investigated."," a"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","Smaller colonies can indicate the spontaneous generation of respiratory-defective petites, whereas larger colonies can indicate the presence of spontaneous mutations that suppress growth defects caused by the desired geno- type."," i"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","Ideally, similar-sized colonies that lack additional genetic changes should be analyzed."," a"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","2. Depending on the expected GCR rate, choose an appropriate volume of YPD liquid medium for growing liquid cultures:"," :DS2aw"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","i. For strains with rates similar to wild type (~10–10 mutations per cell per generation), use 50-mL liquid cultures."," w"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","ii. For moderately increased GCR rates (~10–8 mutations per cell per generation), use 10-mL liquid cultures."," w"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","iii. For very high GCR rates (~10–6 mutations per cell per generation), use 1- to 5-mL liquid cultures."," w"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","iv. For unknown rates, initially try 10-mL liquid cultures."," aw"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","3. For each culture, use a sterile scalpel blade to cut out one colony from the Petri dish. Inoculate each liquid culture with a single colony/agar plug."," :AP1x"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","Ensure that the entire colony is used in the inoculation and that each colony/agar plug is not contaminated with cells from other colonies."," x"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","Grow at least seven cultures for each independent biological isolate; larger numbers of cultures allow more accurate determination of rates."," x"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","4. Grow cultures at 30°C with vigorous shaking until they reach saturation."," :AP1i"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","In rich media, saturation typically occurs after 24-36 h of growth for strains with wild-type growth rates. Sicker strains can take longer to reach saturation and can reach saturation at lower viable cell densities than wild-type strains; determination of experimental growth curves for these types of strains can be helpful."," i"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","5. For each culture, label five sterile microcentrifuge tubes A-E. Add 90 μL of sterile water to each."," :AP1"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","i. Transfer 10 μL of the culture to tube A and vortex."," :AP1"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","ii. Transfer 10 μL of A to B and vortex."," :AP1"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","iii. Transfer 10 μL of B to C and vortex."," :AP1"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","iv. Transfer 10 μL of C to D and vortex."," :AP1"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","v. Transfer 10 μL of D to E and vortex."," :AP1"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","7. Spread all 100 μL of each tube E (i.e., the 10–5 dilution) on 9-cm YPD agar plates."," :AP1e"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","This provides a final effective dilution of 10–6 viable cells per milliliter, relative to the concentration of the culture."," e"
"CSHP-2010-Putnam-pdb.prot5492.txt","2","8. Centrifuge the remainder of the saturated culture at 2000g for 10 min at room temperature. Carefully decant the supernatant."," :AP1"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","9. Resuspend the pellet in 0.2 mL of sterile water per 10 mL of original culture."," :AP1"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","10. Spread the resuspended culture onto 14.5-cm 5-FOA Can plates."," :AP1a"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","For up to 10 mL of culture (~109 cells), one 14.5-cm plate can be used. For larger cultures, divide each culture evenly between multiple plates, plating no more than 109 cells/plate for each culture."," a"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","11. Place the YPD and 5-FOA Can plates in the incubator at 30°C."," :AP1a"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","Colonies with wild-type growth rates should take 3-5 d to appear on 5-FOA Can plates."," a"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","12. Count the number of colonies on the YPD and 5-FOA Can plates."," :AP1ir(t)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","In rich media, wild-type cultures at saturation have ~108 cells/mL, yielding ~100 colonies/YPD plate."," i"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","See Troubleshooting."," r(t)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","13. To calculate the number of viable cells per milliliter (D) for each culture, multiply the number of colonies on the YPD plate by the total dilution factor (in this case, 106; see Steps 6 and 7)."," :AQ1r(6-7)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","see Steps 6 and 7"," r(6-7)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","14. For each culture, calculate the number of viable cells in the culture (N) using the formula N = D × V, where V is the volume of the culture (in milliliters), and D is the viable cells per milliliter (from Step 13)."," :AQ1r(13)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","from Step 13"," r(13)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","15. For each culture, estimate the most likely number of mutational events (m) using the observed number of colonies on the 5-FOA Can plates (r) and the equation m(1.24 + ln[m]) – r = 0."," :AQ1ir(x)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","This equation was originally derived by Lea and Coulson (1949). Values of r as a function of m can readily be tabulated in spreadsheets (Table 1, or m can be calculated from r by iterative methods (e.g., bisection, Newton- Raphson)."," ir(x)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","A Visual Basic macro for calculating m from r is available as Supplemental Material (Suppl_RtoM_Conversion_Macro.doc)."," i"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","16. For each culture, calculate P, the most likely rate (in events per cell per generation) using the for- mula P = m/N, where N is the number of cells in the culture (see Step 14) and m is the most likely number of events (from Step 15)."," :AQ1r(14)r(15)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","see Step 14"," r(14)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","from Step 15"," r(15)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","17. For each biological isolate, order all the most likely rates (P, from Step 16) from smallest to largest, including all zero rates."," :AV1r(16)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","from Step 16"," r(16)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","18. Determine the median rate of the cultures, R."," :AQ3i"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","The median is the value of the middle element in the ordered list (or the average of the two middle values if there are an even number of elements in the list)."," i"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","19. If R = 0, calculate an upper limit for the rate (R) by replacing all cultures with r = 0 with r = 1 and determining the new median of these rates. Report the resulting rate as being less than R. For example, if R = 0 and R = 1.75 × 10–9, the reported rate should be <1.75 × 10–9."," :AQc"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","20. Determine the 95% confidence intervals for the median (see, e.g., http://www.math.unb.ca/ ~knight/utility/MedInt95.htm). For six to 283 cultures, calculate the lower (L) and upper (U) 95% confidence interval limits using the equations L = floor[(M+1) / 2 – 0.9789 M ] and U = (M + 1) – L, where M is the number of cultures."," :AV1; AQ1; AV1r(t)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","For six to 283 cultures, calculate the lower (L) and upper (U) 95% confidence interval limits using the equations L = floor[(M+1) / 2 – 0.9789 M ] and U = (M + 1) – L, where M is the number of cultures."," AQ1"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","Briefly, the lower and upper 95% confidence interval limits correspond to the L-th and U-th posi- tions in the ordered list of individual rates. For example, if the number of cultures (M) 14, L = floor[15 / 2 – 0.9789 14] = 3; U = (14 + 1) – 3 = 12. Thus, the 95% confidence interval is defined by the third and 12th lowest mutation rates."," AV1r(t)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","See Troubleshooting."," r(t)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","21. If the median rates for the different biological isolates are within the 95% confidence intervals, merge all rates from the different biological isolates. Determine the median rate and the 95% con- fidence intervals for the combined list of rates (i.e., repeat Steps 17-20 for the entire set of rates from all biological isolates of a given genotype)."," :AV1o; CL(17-20)"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","If the median rates for the different biological isolates are within the 95% confidence"," o"
"CSHP-2010-Putnam-pdb.prot5492.txt","3","repeat Steps 17-20 for the entire set of rates from all biological isolates of a given genotype"," CL1(17-20)"
"CSHP-2010-Song-pdb.prot5384.txt","3","1. Centrifuge 50 million human or other mammalian cells in single-cell suspension at 900 rpm for 5 min in 50-mL tubes. Use vacuum aspiration or a pipette to carefully remove the supernatant (do not pour off liquid). Flick to resuspend the cell pellet in residual liquid."," :AP1r(Troubleshooting)x"
"CSHP-2010-Song-pdb.prot5384.txt","3","Because the pellet is loose, leave a 1 mm-thick supernatant layer over the pellet during washes to reduce cell loss."," x"
"CSHP-2010-Song-pdb.prot5384.txt","3","See Troubleshooting."," r(Troubleshooting)"
"CSHP-2010-Song-pdb.prot5384.txt","3","2. Wash the cells twice with cold 1X PBS (50 mL of PBS for each wash). If starting with two or more tubes of cell culture, combine cells into one tube for the first wash."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","3","3. Resuspend the final cell pellet in 500 μL of cold RSB buffer by gentle flicking and transfer to a 15-mL conical tube (keep on ice). Rinse the 50-mL tube with 500 μL of cold RSB buffer to collect any remaining cells, and combine the rinse with the first 500 μL of cell suspension."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","3","4. Slowly pour 14 mL of lysis buffer (cold RSB buffer + 0.1% IGEPAL CA-630) into the 15-mL tube containing the cell suspension. Invert five to 10 times. Make sure there is no detectable precipitation at this step."," :AP1iwx"
"CSHP-2010-Song-pdb.prot5384.txt","3","(cold RSB buffer + 0.1% IGEPAL CA-630)"," i"
"CSHP-2010-Song-pdb.prot5384.txt","3","Make sure there is no detectable precipitation at this step."," x"
"CSHP-2010-Song-pdb.prot5384.txt","3","Invert five to 10 times."," w"
"CSHP-2010-Song-pdb.prot5384.txt","3","5. Check cells after lysis by staining with Trypan blue and examining under a microscope."," :AP1ir(Troubleshooting)"
"CSHP-2010-Song-pdb.prot5384.txt","3","If lysis has been effective, at least 99% of the cells should be stained."," i"
"CSHP-2010-Song-pdb.prot5384.txt","3","See Troubleshooting."," r(Troubleshooting)"
"CSHP-2010-Song-pdb.prot5384.txt","4","6. Centrifuge immediately at 500g for 10 min at 4°C to pellet nuclei, and remove supernatant completely. Do not leave any supernatant on the pellet."," :AP1x, CB(7)"
"CSHP-2010-Song-pdb.prot5384.txt","4","Do not leave any supernatant on the pellet."," x"
"CSHP-2010-Song-pdb.prot5384.txt","4","Proceed with Step 7 during centrifugation."," CB(7)"
"CSHP-2010-Song-pdb.prot5384.txt","4","In the following steps, be sure to follow the instructions for mixing (e.g., if the directions specify “flick” or “invert,” do not vortex or pipette the material)."," :X1x"
"CSHP-2010-Song-pdb.prot5384.txt","4","i. Use 1X DNase incubation buffer to make five concentrations (0.01, 0.03, 0.1, 0.3, and 1 U/μL) of recombinant DNase I in five microcentrifuge tubes (1.5-mL); keep tubes on ice."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","4","When diluting DNase, make sure dilutions are extensively mixed."," x"
"CSHP-2010-Song-pdb.prot5384.txt","4","ii. Label seven empty microcentrifuge tubes #1-#7 and place on ice."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","4","iii. Pipette 12 μL of the indicated concentration of DNase into each of tubes #3-#7. Do not add DNase to tubes #1 and #2."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","4","Do not add DNase to tubes #1 and #2."," x"
"CSHP-2010-Song-pdb.prot5384.txt","5","8. Suspend nuclei (lysed cells) in 840 μL of cold 1X DNase incubation buffer and mix by flicking. The pellet should appear white and fluffy and should be suspended completely."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","5","9. Slowly pipette 120 μL of nuclei suspension into tubes #1-#7 by using wide-bore pipette tips. Gently swirl to resuspend nuclei before each transfer."," :AP1m"
"CSHP-2010-Song-pdb.prot5384.txt","5","10. Keep tube #1 on ice, and incubate tubes #2-#7 for 15 min in a 37°C water bath."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","5","11. Melt several tubes of InCert low-melt agarose at 75°C. Once they are completely melted, transfer to 55°C."," :AP1r(13)"
"CSHP-2010-Song-pdb.prot5384.txt","5","Agarose will be used in Step 13."," r(13)"
"CSHP-2010-Song-pdb.prot5384.txt","5","12. Slowly pipette 330 μL of 50 mM EDTA into each of tubes #1-#7 to stop the reactions, and invert tubes five times to mix. Keep tubes at room temperature until all have been mixed, then equilibrate for 1 min at 55°C."," :AP1m"
"CSHP-2010-Song-pdb.prot5384.txt","5","into each of tubes #1-#7"," m"
"CSHP-2010-Song-pdb.prot5384.txt","5","13. Slowly pipette 450 μL of InCert low-melt agarose (from Step 11) into each tube. Invert four times to mix."," :AP1mr(11)"
"CSHP-2010-Song-pdb.prot5384.txt","5","from Step 11"," r(11)"
"CSHP-2010-Song-pdb.prot5384.txt","5","into each tube"," m"
"CSHP-2010-Song-pdb.prot5384.txt","5","14. Pipette mixtures into CHEF disposable plug molds using wide-bore pipette tips. Let sit for ~5 min at 4°C to solidify. The volume of each plug is ~80 μL."," :AP1i"
"CSHP-2010-Song-pdb.prot5384.txt","5","Low-melt gel agarose plugs are used to stabilize high-molecular-weight DNA and protect against random shearing."," i"
"CSHP-2010-Song-pdb.prot5384.txt","5","15. Carefully release plugs into 50 mL of LIDS buffer in a 50-mL conical tube, place a green screened plug cap (for washing plugs) between each tube and cap, and shake horizontally at 50-60 rpm for 1-2 h at room temperature. Keep plugs with different DNase1 concentrations in separate conical tubes."," :AP1w"
"CSHP-2010-Song-pdb.prot5384.txt","5","50-60 rpm for 1-2 h"," w"
"CSHP-2010-Song-pdb.prot5384.txt","5","16. Replace with fresh LIDS buffer and incubate tubes overnight at 37°C, horizontal without shaking."," :AP1a"
"CSHP-2010-Song-pdb.prot5384.txt","5","horizontal without shaking."," a"
"CSHP-2010-Song-pdb.prot5384.txt","5","17. Wash plugs five times with 50 mL of 50 mM EDTA per tube, 1 h each wash, shaking at 60 rpm at room temperature. Prior to the fourth and fifth EDTA washes, wash the filters and lids with water to remove residual detergent from the filter and lid threads. Make sure that no detergent bubbles are left in the tubes after the fifth washing."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","5","Make sure that no detergent bubbles are left in the tubes after the fifth washing."," x"
"CSHP-2010-Song-pdb.prot5384.txt","5","18. Store the plugs at 4°C in 50 mM EDTA."," :AP1i"
"CSHP-2010-Song-pdb.prot5384.txt","5","Plugs can be stored indefinitely under these conditions."," i"
"CSHP-2010-Song-pdb.prot5384.txt","5","i. Prepare a 1% UltraPure Agarose gel in 0.5X TBE."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","5","ii. Load one-third of a plug from each DNase concentration into the gel by sliding each plug piece into a separate well using two metal spatulas."," :AP1a"
"CSHP-2010-Song-pdb.prot5384.txt","5","using two metal spatulas."," a"
"CSHP-2010-Song-pdb.prot5384.txt","5","iii. Use yeast chromosome PFG marker to size smearing patterns."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","5","iv. Set PFG unit to keep 0.5X TBE running buffer chilled at 16°C."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","5","v. Set running parameters as follows: 20-60 sec switch time; 18 h running time; 6 V/cm (180 V total)."," :AP1w"
"CSHP-2010-Song-pdb.prot5384.txt","5","20-60 sec switch time"," w"
"CSHP-2010-Song-pdb.prot5384.txt","5","20. Compare gel pattern to Figure 2. Optimal amounts for DNase-seq are 0.4 U, 1.2 U, and 4.0 U."," :AP1aei, AP4r(Troubleshooting)"
"CSHP-2010-Song-pdb.prot5384.txt","5","Optimal amounts for DNase-seq are 0.4 U, 1.2 U, and 4.0 U."," e"
"CSHP-2010-Song-pdb.prot5384.txt","5","The smearing patterns (50-100 kb to 1 Mb in size) seen in the lanes for the 0.4 U, 1.2 U, and 4.0 U DNase amounts are usually ideal for making DNase-seq libraries from mammalian cells. Because DNase HS sites are"," i"
"CSHP-2010-Song-pdb.prot5384.txt","6","We have found that using more heavily digested DNA results in lower signal to noise."," a"
"CSHP-2010-Song-pdb.prot5384.txt","6","DNase concentrations to achieve optimal smearing sizes may differ for each cell line and therefore must be determined empirically for each cell type. Use of this protocol on smaller eukaryotic genomes (such as yeast) may require different optimal smearing patterns."," AP4r(Troubleshooting)"
"CSHP-2010-Song-pdb.prot5384.txt","6","See Troubleshooting."," r(Troubleshooting)"
"CSHP-2010-Song-pdb.prot5384.txt","6","need to be blunt-ended by T4 DNA polymerase before ligating"," x"
"CSHP-2010-Song-pdb.prot5384.txt","6","Because DNase I nicks DNA and leaves single-strand overhangs, these overhangs need to be blunt-ended by T4 DNA polymerase before ligating to blunt-ended linker 1."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","6","1. Wash the EDTA-soaked plugs twice with 50 mL of 1X NEB buffer 2 per tube, 1 h for each wash, shaking at 60 rpm at room temperature."," :AP1i"
"CSHP-2010-Song-pdb.prot5384.txt","6","This step removes EDTA from the plugs."," i"
"CSHP-2010-Song-pdb.prot5384.txt","6","22. Remove all traces of liquid from the 50-mL conical tubes and push plugs to the bottom of the tubes."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","6","i. Mix the plugs in the 50-mL conical tubes with T4 DNA polymerase and other reagents as indicated."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","6","ii. Incubate for 4 h at room temperature. Do not mix plugs from different optimal DNase concentrations (they will be combined in Step 29)."," :AP1r(29)"
"CSHP-2010-Song-pdb.prot5384.txt","6","they will be combined in Step 29)"," r(29)"
"CSHP-2010-Song-pdb.prot5384.txt","6","iii. Shake the reactions occasionally."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","6","24. Rinse the plugs quickly with 1X TE buffer to remove residual enzyme."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","i. Transfer plugs to 1.5-mL microcentrifuge tubes. Add 500 μL of 1X TE buffer and heat for 15 min at 65°C to melt. Flick every 5 min to make sure agarose is dissolving."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","ii. Extract each tube using equal volumes of a series of phenol, phenol:chloroform:isoamyl alcohol, and chloroform."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","iii. Precipitate the DNA with 1 μL of 20 mg/mL glycogen, 50 μL of 3 M NaOAc, and 1.0 mL of ethanol."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","26. Pellet the DNA by centrifuging at 16,000g for 10-15 min at 4°C, wash the pellet with two volumes of 70% ethanol, centrifuge again, and remove all residual liquid. Let dry for <4 min."," :AP1a"
"CSHP-2010-Song-pdb.prot5384.txt","7","Longer drying times may cause difficulty in dissolving the DNA pellet."," a"
"CSHP-2010-Song-pdb.prot5384.txt","7","27. Resuspend the DNA pellet in 40 μL of 10 mM Tris-Cl (pH 8.0)."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","Stock linkers are at a concentration of 25 pmol/μL."," i"
"CSHP-2010-Song-pdb.prot5384.txt","7","i. Anneal oligo 1a and 1b (to make linker 1) and oligo 2a and 2b (to make linker 2) in 1X NEB buffer 2."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","ii. Heat for 5 min at 95°C."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","iii. Slowly cool to room temperature. Make fresh or store aliquots at −20°C."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","i. Combine reagents as indicated."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","ii. Incubate tubes overnight in a thermal cycler at 20°C."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","i. Prepare a 0.8% UltraPure LMP Agarose gel using 10 mM EDTA and 1X TBE."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","ii. Run the reactions from Step 29 for 30-50 min at 80 V in 10 mM EDTA and 1X TBE running buffer."," :AP1r(29)w"
"CSHP-2010-Song-pdb.prot5384.txt","7","from Step 29"," r(29)"
"CSHP-2010-Song-pdb.prot5384.txt","7","for 30-50 min"," w"
"CSHP-2010-Song-pdb.prot5384.txt","7","i. Cut out the high-molecular-weight smear (leave the linker-only band behind)."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","7","leave the linker-only band behind"," x"
"CSHP-2010-Song-pdb.prot5384.txt","7","ii. Put the gel into 500 μL of 1X TE and heat for 15 min at 65°C to melt. Gently flick every few minutes to make sure agarose is dissolving."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","iii. Extract with equal volumes of a series of phenol, phenol:chloroform:isoamyl alcohol, and chloroform."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","iv. Precipitate the DNA with 1 μL of 20 mg/mL glycogen, 50 μL of 3 M NaOAc, and 1 mL of ethanol."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","v. Wash the DNA pellet with two volumes of 70% ethanol."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","7","vi. Dry the pellet and resuspend the DNA in 75 μL of water."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","i. Prepare the following mixture."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","ii. Incubate the reaction for 1.5 h at 37°C."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","i. Extract the digestion with equal volumes of phenol:chloroform:isoamyl alcohol and chloroform."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","ii. Precipitate the DNA by adding 1 μL of 20 mg/mL glycogen, 10 μL of 3 M NaOAc, and 325 μL of ethanol."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","iii. Wash the pellet with 70% ethanol."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","iv. Dry the pellet and resuspend it in 50 μL of water."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","Use magnetic stand and vacuum aspiration for all wash steps."," x"
"CSHP-2010-Song-pdb.prot5384.txt","8","i. Wash the beads twice with 1 mL of 1X TE."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","8","ii. Wash the beads once with 1 mL of 1X B&W buffer. Remove wash buffer before beginning the next step."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","8","Remove wash buffer before beginning the next step."," x"
"CSHP-2010-Song-pdb.prot5384.txt","8","i. Suspend the beads in a mixture of 50 μL of MmeI-digested DNA and 50 μL of 2X B&W buffer."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","ii. Incubate for 30 min at 30°C, flicking the tubes every 5 min to resuspend the beads."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","iii. Remove the supernatant and wash the beads five times with 1 mL of 1X TE."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","iv. Wash the beads once with 100 μL of 1X ligation buffer. Remove the wash buffer before the next step."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","8","Remove the wash buffer before the next step."," x"
"CSHP-2010-Song-pdb.prot5384.txt","8","i. Add the following reagents to the Dynal beads (~10 μL)."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","ii. Rotate the tube for 4 h at room temperature."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","i. Wash the beads once with 1 mL of 1X TE."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","8","ii. Add 500 μL of 0.15 M NaOH directly to the beads."," :AP1i"
"CSHP-2010-Song-pdb.prot5384.txt","8","This step denatures double-stranded DNA template on the beads and removes the nonbiotinylated strand to eliminate false-positive sequencing signals from mismatched base pairs at the end of linker 2."," i"
"CSHP-2010-Song-pdb.prot5384.txt","8","iii. Rotate the tube for 5 min at room temperature."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","iv. Wash the beads five times with 1 mL of 1X TE."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","v. Resuspend the beads in 25 μL of 10 mM Tris-Cl (pH 8.0)."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","i. Prepare the following mixture."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","ii. Denature for 30 sec at 98°C, followed by 12 amplification cycles (10 sec, 98°C; 30 sec, 60°C; 15 sec, 72°C), followed by extension for 7 min at 72°C."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","i. Load 1 μL of the 25-bp DNA ladder into one well and 50 μL of PCR products from Step 38 into two adjoining wells of a 4%-20% TBE PAGE gel."," :AP1r(38)"
"CSHP-2010-Song-pdb.prot5384.txt","9","from Step 38"," r(38)"
"CSHP-2010-Song-pdb.prot5384.txt","9","ii. Electrophorese for 2 h at 120 V."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","iii. Pry apart the cassette and stain the gel in 1X TE + 0.5 μg/mL ethidium bromide in a clean container for 2-3 min."," :AP1r(Troubleshooting)vw"
"CSHP-2010-Song-pdb.prot5384.txt","9","for 2-3 min"," w"
"CSHP-2010-Song-pdb.prot5384.txt","9","A representative example of amplification is shown in Figure 3."," v"
"CSHP-2010-Song-pdb.prot5384.txt","9","See Troubleshooting."," r(Troubleshooting)"
"CSHP-2010-Song-pdb.prot5384.txt","9","i. Cut out the 86-bp band (containing linkers + insert) with a clean razor blade."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","9","with a clean razor blade."," x"
"CSHP-2010-Song-pdb.prot5384.txt","9","ii. Place the gel slice into a 0.5-mL microcentrifuge tube whose bottom has been punctured by a 21-gauge needle."," :AP1x"
"CSHP-2010-Song-pdb.prot5384.txt","9","by a 21-gauge needle"," x"
"CSHP-2010-Song-pdb.prot5384.txt","9","iii. Set this tube into a 1.5-mL round-bottom microcentrifuge tube."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","iv. Centrifuge the gel slice through the hole into the 1.5-mL tube, in a microcentrifuge at full speed for 2 min."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","i. Add 100 μL of 1X NEB buffer 2 to the gel."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","ii. Elute the DNA by rotating the tube gently for 2 h at room temperature."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","9","iii. Transfer the eluate and the gel debris onto the top of a Spin-X filter. Centrifuge the filter in a microcentrifuge at full speed for 2 min to remove traces of gel."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","10","i. Add 1 μL of 20 mg/mL glycogen, 10 μL of 3 M NaOAc, and 325 μL of ethanol. Precipitate the DNA for 30 min at −20°C."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","10","ii. Dry the DNA pellet in the air for 5 min."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","10","iii. Resuspend the pellet in 10 μL of 1X TE."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","10","i. Run 1 μL of recovered DNA on a 4%-20% TBE gel."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","10","ii. Stain the gel with ethidium bromide."," :AP1"
"CSHP-2010-Song-pdb.prot5384.txt","10","44. Sequence libraries on a Solexa/Illumina DNA sequencer using the custom sequencing primer."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","3","Because experimental designs may vary substantially (see Szyjka et. al. [2008]; Knott et al. [2009a]), we describe only the addition of BrdU to the growth medium and cell harvesting. For the reference DNA sample (to be fluorescently labeled and cohybridized onto the microarray with the IP DNA), we typically use genomic DNA prepared from G1-arrested cells (not labeled with BrdU). However, BrdU-labeled DNA may be used, and DNA from different cell cycle stages or time points may be used. For the former, genomic DNA from 10-20 mL of G1-arrested cells (OD600~ 0.5-1.0) should be prepared (Steps 3-22) in parallel with BrdU-incorporated genomic DNA. For the latter, 1 ug of BrdU-incorporated genomic DNA obtained at Step 20 is used. In both cases, reference DNA should be amplified to account for potential differences in amplification efficiencies of different sequences."," :X1air(20)r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","3","see Szyjka et. al. [2008]; Knott et al. [2009a]"," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","3","reference DNA should be amplified to account for potential differences in amplification efficiencies of different sequences."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","3","obtained at Step 20"," r(20)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","3","1. Grow 10-20 mL of S. cerevisiae cells to an OD600 ~ 0.5-1.0 in YEPD (or other) medium."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","incubate for the desired period. For very short incubations with BrdU (e.g., 10-15 min), doubling the concentration of BrdU may be helpful."," AP1a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","For very short incubations with BrdU (e.g., 10-15 min), doubling the concentration of BrdU may be helpful."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","2. Add BrdU to a final concentration of 400 μg/mL and incubate for the desired period. For very short incubations with BrdU (e.g., 10-15 min), doubling the concentration of BrdU may be helpful."," :AP2; AP1a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","transfer the culture to a 15- or 50-mL conical screw-cap tube."," AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","3. To harvest, add sodium azide to 0.1% final concentration and transfer the culture to a 15- or 50-mL conical screw-cap tube."," :AP2; AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","4. Pellet the cells by centrifugation in a swinging-bucket rotor at 1500g for 5 min at 4°C. Discard the supernatant."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","5. Resuspend the cell pellet by pipetting up and down in 20 mL of ice-cold TBS for yeast. Pellet the cells by centrifugation at 1500g for 5 min at 4°C."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","6. Resuspend the cell pellet in 1 mL of ice-cold TBS for yeast with a micropipettor and transfer to a chilled, 2-mL microcentrifuge tube with a gasket-sealed screw-cap."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","Remove the supernatant without disturbing the cell pellet."," x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","7. Pellet the cells in a microcentrifuge at full speed (~16,000g) for a few seconds. Remove the supernatant without disturbing the cell pellet."," :APax"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","At this point, the cell pellet may be frozen rapidly using a dry ice-ethanol or liquid nitrogen bath and stored at −20°C."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","resuspend the cells in 500 μL of lysis buffer for genomic DNA."," AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","8. Thaw the cell pellet (if necessary) and resuspend the cells in 500 μL of lysis buffer for genomic DNA."," :AP1o; AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","if necessary"," o"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","9. Add an equal volume (~0.6 mL) of glass beads to the cell suspension."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","10. Tightly cap the tube and place it into a FastPrep instrument. Run the FastPrep at power setting 5.5 for 45 sec."," :AP1a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","Other vortexers may be used, but specific settings to achieve maximal cell breakage will vary."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","11. Remove the tube from the FastPrep and centrifuge at full speed for a few seconds to collapse any foam. Add 25 μL of 5 M NaCl to each sample."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","12. Repeat Step 10."," :CL(10)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","i. Invert the tube and flick to knock the beads and solution away from the bottom of the tube."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","attached to a small syringe to hold the needle safely"," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","ii. Puncture the bottom of the tube twice with a red-hot 26-gauge needle (attached to a small syringe to hold the needle safely)."," :AP1ax"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","with a red-hot 26-gauge needle"," x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","iii. Immediatelyinsertthetubeintoa5-mLpolypropylenesnap-captubewithitscapremoved (it may only fit partially)."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","iv. Centrifuge in a swinging-bucket rotor at 350g for 2 min to collect the lysate."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","v. Remove the tube combination from the centrifuge."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","vi. Remove the FastPrep tube, which should contain only the beads, from the 5-mL tube, which contains the cell lysate."," :AP1i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","The lysate is partially pelleted at this point."," i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","14. Decant the soluble lysate to a fresh microcentrifuge tube and leave behind the pellet, which is mainly cell debris (some carryover is acceptable)."," :AP1w"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","some carryover is acceptable"," w"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","i. Insert the microtip horn of a sonicator about halfway down the depth of the lysate in the microcentrifuge tube."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","ii. Sonicate for 20 sec with constant output on low power. With the Branson 250 sonicator (with microtip attachment), use power setting 2.0 and 100% duty cycle."," :AP1ar(Troubleshooting)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","iii. Chill the sample on ice between rounds of sonication."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","4","Specific settings must be determined if a different sonicator is used."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","See Troubleshooting."," r(t)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","16. Repeat Step 15 (multiple times if smaller DNA shear size is desired)."," :CL(15), AI2"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","i. Add 500 μL of PCI to each sample and vortex vigorously."," :AP1m"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","to each sample"," m"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","ii. Separate the phases by centrifugation at full speed (~16,000g) for 5 min."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","iii. Transfer the aqueous phase (~450 μL) to a new microcentrifuge tube."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","18. Repeat Step 17."," :CL(17)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","19. Add 1 mL of ethanol to each sample, mix, and centrifuge at full speed (~16,000g) for 15 min. Decant the supernatant and allow residual liquid to evaporate."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","20. Resuspend the pellet in 100 μL of TE (pH 7.6) + 1 μL of RNaseA and incubate for 30 min at 37°C."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","21. Purify the samples using a QIAquick PCR Purification Kit according to the manufacturer’s instructions. Elute with 50 μL of TE (pH 7.6)."," :AP1r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","according to the manufacturer’s instructions"," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","22. Measure the DNA concentration using a NanoDrop spectrophotometer or similar device."," :AP1ax"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","At least 2.5 μg (as measured with NanoDrop) of genomic DNA should be recovered from a 20-mL cell culture with OD ~ 1."," x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","Samples may be stored overnight at 4°C or for a few weeks at -20°C."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","In these steps, BrdU-labeled genomic DNA that has been isolated and sheared is denatured and immunoprecipitated using an antibody specific to BrdU. Reference DNA is not subjected to this procedure and is set aside until the amplification in Steps 35-38. If immunoprecipitated DNA is to be analyzed with high-throughput sequencing, we do not recommend a blocking step (see below), because it uses nonhomologous DNA that may remain in the immuno- precipitated sample and complicate sequencing analysis. Instead, we suggest the use of Dynabeads Protein G, which does not require blocking."," :X1ar(35-38)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","we do not recommend a blocking step (see below), because it uses nonhomologous DNA that may remain in the immuno- precipitated sample and complicate sequencing analysis. Instead, we suggest the use of Dynabeads Protein G, which does not require blocking."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","in Steps 35-38."," r(35-38)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","23. Combine in a microcentrifuge tube:"," :AP1a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","If different amounts of DNA are used, alter the amounts and volumes appropriately, maintaining all concentrations."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","i. Heat the sample on a heat block for 10 min at 95°C to denature the DNA."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","ii. Snap-cool the sample in an ice-water bath for a few minutes."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","iii. Briefly centrifuge the tube in a microcentrifuge to collect the solution in the bottom of the tube."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","i. Dilute the appropriate amount (e.g., 1:1000) of anti-BrdU antibody in ice-cold IP buffer for genomic DNA."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","ii. Add 200 μL of diluted antibody to the denatured DNA sample."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","iii. Incubate with gentle agitation (e.g., using a tube rotator) for 1 h (up to overnight) at 4°C."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","5","26. Briefly centrifuge to collect the sample in the bottom of the tube. Using a wide-bore pipette tip, add 30 μL of a 50% suspension of Protein G-Sepharose beads in PBS for genomic DNA and incubate on a tube rotator for 1 h at 4°C."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","27. Gently pellet the beads using a microcentrifuge at ~800g for 1 min at room temperature. Carefully remove the supernatant, avoiding the beads."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","28. Add 1 mL of ice-cold IP buffer for genomic DNA and place the sample on a tube rotator for ~3-5 min at room temperature. Repeat Step 27."," :AP1w; CL(27)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","for ~3-5 min"," w"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","Repeat Step 27."," CL(27)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","29. Repeat Step 28 two additional times."," :CL(28); CL(28)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","Repeat Step 27."," CL(27)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","30. Add 1 mL of TE (pH 7.6) and place on a tube rotator for ~3-5 min at room temperature. Repeat Step 27."," :AP1w; CL(27)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","on a tube rotator for ~3-5 min"," w"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","31. Add 100 μL of elution buffer for genomic DNA to the beads and incubate in a heat block for 15 min at 65°C."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","32. Pellet the beads as in Step 27. Transfer the eluate to a fresh microcentrifuge tube."," :AP1r(27)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","as in Step 27"," r(27)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","33. Purify the eluate using a MinElute PCR Purification Kit according to the manufacturer’s protocol with the following modifications:"," :AP1r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","according to the manufacturer’s protocol"," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","i. Perform two washes with PE Buffer."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","ii. Elute with 11 μL of 0.2X EB prewarmed to ~50°C."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","or for a few weeks at -20°C. For longer-term storage, dry the DNA samples and place at -20°C or -80°C."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","34. Store samples overnight at 4°C, or for a few weeks at -20°C. For longer-term storage, dry the DNA samples and place at -20°C or -80°C."," :AP1ar(t)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","At this point, you may proceed to the DNA amplification. However, before investing time and resources into these steps, it is advisable to confirm the expected level of enrichment (if possible) by direct PCR analysis."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","See Troubleshooting."," r(t)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","To generate sufficient DNA for analysis, the IP and reference DNA samples must be amplified before labeling the DNA with a fluorophore. Commercially available kits are available for efficiently amplifying sheared DNA fragments."," :AP2"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","35. Determine the DNA concentration of the IP and reference samples using a NanoDrop or similar instrument. Approximately 50 ng (as measured by NanoDrop) of IP DNA should be recovered."," :AQ3r(Troubleshooting)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","See Troubleshooting."," r(Troubleshooting)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","36. Use the GenomePlex Complete WGA Kit to separately amplify 10 ng of IP and 10 ng of reference DNA according to the manufacturer’s protocol."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","37. After the amplification reaction, purify the DNA samples using a QIAquick PCR Purification Kit according to the manufacturer’s instructions. Elute using 30 μL of buffer EB."," :AP1r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","according to the manufacturer’s instructions"," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","More than 3 μg (as measured by NanoDrop) of WGA-amplified DNA should be recovered."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","38. Measure the concentration of each sample with a NanoDrop. More than 3 μg (as measured by NanoDrop) of WGA-amplified DNA should be recovered."," :AQ3ar(t)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","At this point, the samples may be stored at -20°C, or dried for long-term storage."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","See Troubleshooting."," r(t)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","There are various methods for fluorescently labeling DNA samples using fluorophores. This section describes the direct labeling of samples by a Klenow (exo-) extension reaction using Cy-labeled random 9-mers as primers. Always protect Cy dyes and Cy-labeled DNA samples from excessive light exposure."," :X1ax"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","Always protect Cy dyes and Cy-labeled DNA samples from excessive light exposure."," x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","39. Use the NimbleGen Dual-Color DNA Labeling Kit (or similar) to label the IP DNA with Cy5 and reference DNA with Cy3 according to the manufacturer’s instructions."," :AP1air(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","according to the manufacturer’s instructions."," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","The Klenow extension reaction in this step further amplifies the DNA samples. This amplification is dependent on several factors, including the Cy-conjugated random 9-mer primer concentration and the amount of Klenow used."," i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","6","We have found that hybridizations can be performed using less labeled DNA without significantly affecting the data quality; therefore, if desired, in this step both Klenow enzyme and primers can be conserved. However, scaling back on these reagents results in lower amounts of amplified DNA. We have found that a typical reaction to label 1 μg of DNA using primers at OD = 1.0 and 100 U of Klenow produces >20 μg of labeled DNA; using primers at OD = 0.1 and 100 U of Klenow produces ~5 μg of DNA; using primers at OD = 0.1 and 50 U of Klenow produces ~3 μg of DNA."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","according to the manufacturer’s protocol"," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","40. Purify the labeled DNA using a MinElute PCR Purification Kit according to the manufacturer’s protocol. Elute with 10 μL of 10 mM EDTA."," :AP1ar(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","The volume used to dissolve the labeled DNA can be adjusted depending on the anticipated amount of DNA produced in the labeling reaction."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","41. Determine the DNA concentration of the Cy-labeled IP and reference samples using a NanoDrop."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","42. In a new microcentrifuge tube, for each sample, combine equal amounts of Cy5-labeled IP DNA and Cy3-labeled reference DNA. Dry the combined DNA using a vacuum concentrator."," :AP1ar(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","We typically combine 1-2 μg of IP and reference DNA for hybridization onto NimbleGen yeast arrays; for larger genomes, more DNA should be hybridized."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","The amounts required for hybridization may vary."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","Follow the array manufacturer’s guidelines."," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","43. Proceed to the hybridization or store the samples at −20°C, protected from light."," :CL(44) | AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","or store the samples at −20°C, protected from light."," AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","This section describes the hybridization of DNA onto NimbleGen high-density oligonucleotide tiling microarrays representing the yeast genome. Adapted from the protocols supplied by NimbleGen, it uses NimbleGen reagents with standard hybridization chambers and equipment. For other array configurations or platforms, hybridization supplies and protocols included by the microarray manufacturer should be used. For customized microarrays, hybridization temperature and buffers may need to be optimized depending on the array design and slide-surface chemistry."," :X1i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","44. Dissolve the combined IP and reference DNA with 9.5 μL of H2O."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","45. Prepare the hybridization chambers by coating the gaskets with a thin layer of vacuum grease. Clean the inside of each chamber with ethanol and, if desired, compressed air."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","46. Using components from a NimbleGen Hybridization Kit, prepare the following hybridization mix for each sample:"," :AP1aim"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","for each sample"," m"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","For hybridizations requiring different volumes of sample, adjust the volumes above accordingly, maintaining the concentrations."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","We describe the hybridization using 22- × 22-mm LifterSlips with a capacity of ~40 μL."," i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","47. Denature a sample in a heat block for 5 min at 95°C, protected from light."," :AP1ar(47-52)w"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","Alternatively, multiple samples can be denatured together and then placed on a heat block at 42°C until they are ready to be applied to each microarray."," w"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","Steps 47-52"," r(47-52)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","typically carried out one sample at a time."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","48. While the sample is denaturing, insert a microarray slide into the bottom portion of a hybridization chamber. Place the hybridization chamber and slide on an inverted heat block at 55°C to prewarm the slide before hybridization."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","49. Use compressed air to remove any dust from a LifterSlip. Gently place the LifterSlip onto the prewarmed slide, chamber side toward the glass slide, completely covering the array features area."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","50. Quickly centrifuge the denatured sample for a few seconds to collect material in the bottom of the tube. Apply the entire sample to the slide by pipetting slowly along an open edge of the LifterSlip while allowing capillary action to draw the sample under the LifterSlip."," :AP1x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","Avoid introducing air bubbles and disrupting the LifterSlip position."," x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","51. Add 15 μL of 3X SSC buffer to each of the two small wells in the bottom portion of the hybridization chamber."," :AP1i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","The 3X SSC will keep the chamber humidified during hybridization."," i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","7","52. Close the hybridization chamber and incubate in a water bath or hybridization oven in the dark at 42°C. Incubate for 12 to 20 h, typically overnight."," :AP1"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","53. Wash and dry the slides using the protocol, solutions, slide racks, and wash chambers found in the NimbleGen Wash Buffer Kit and the NimbleGen Array Processing Accessories Kit. For other array platforms, perform washes as directed by the microarray supplier."," :AP1ar(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","protocol"," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","For customized microarrays, wash buffers may need to be optimized, depending on the array design and slide- surface chemistry."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","54. Store dried slides in a slide box, protected from light, and preferably in an ozone-free environment. Scan slides as soon as possible."," :AP1x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","Scan slides as soon as possible."," x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","55. Scan slides according to the description in the NimbleGen Array’s User’s Guide. Save the 532- and 635-nm images as single image .tif files."," :AP1r(t)r(x)x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","Save the 532- and 635-nm images as single image .tif files."," x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","in the NimbleGen Array’s User’s Guide"," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","See Troubleshooting."," r(t)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","56. Using NimbleScan software, open the scanned image files and create a pair report, as described in the User’s Guide. The pair report generates a GFF file type that can be analyzed further and plotted along chromosomal coordinates using NimbleScan software."," :AV1r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","as described in the User’s Guide."," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","57. Arrays can be “stripped” and reused with the Nimblegen Array Reuse Kit. However, these arrays show decreases in their M = log(IP/Total) signals with each successive array use (AU). This can cause false positives when attempting to detect variations between experiments whose corresponding AUs differ. To minimize this potential bias, perform comparisons with microarrays subjected to equal numbers of AUs."," :X1aiox"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","However, these arrays show decreases in their M = log(IP/Total) signals with each successive array use (AU)."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","can be “stripped” and reused"," o"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","his can cause false positives when attempting to detect variations between experiments whose corresponding AUs differ."," i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","To minimize this potential bias, perform comparisons with microarrays subjected to equal numbers of AUs."," x"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","58. Following array scanning and Cy3 and Cy5 signal detection, perform array normalization to remove the biases that are typically seen in microarray data."," :AV1air(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","The characteristics of BrdU-IP-chip experiments render traditional normalization procedures (for RNA-chip and ChIP-chip) suboptimal,"," i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","we have designed and implemented normalization software specifically for these data sets (Knott et al. 2009b)."," r(x)"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","Also, we do not recommend traditional ChIP-chip enrichment detection methods for identifying BrdU-enriched genomic regions."," a"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","These methods typically assume a bimodal M-distribution, which is not the case in BrdU-IP-chip data sets."," i"
"CSHP-2010-Viggiani-pdb.prot5385.txt","8","we have developed an enrichment detection method specifically for these data sets (Knott et al. 2009b)."," r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","Before investigating valuable samples, beginning investigators should practice making control libraries with some avail- able total RNA to develop their abilities to check cDNA synthesis by real-time PCR, to visualize semisuppressive PCR smears on gels, and to interpret Bioanalyzer profiles of prepared libraries."," :X1a"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","This process produces first-strand cDNAs from total RNA by random priming; the 5 ends are captured through template switching. Perform all procedures in RNase-free conditions on a workbench dedicated only to RNA work."," :X1ix"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","This process produces first-strand cDNAs from total RNA by random priming; the 5 ends are captured through template switching."," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","Perform all procedures in RNase-free conditions on a workbench dedicated only to RNA work."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","1. Mix 8 μL of sorbitol/trehalose stock solution, 1 μL of 100 μM reverse transcription primer, and 1 μL of 1 mM template-switching DNA/RNA oligonucleotide."," :AP1ix"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","Sorbitol/trehalose solutions are viscous"," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","mix very carefully by pipetting at least 10 times."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","2. Aliquot 1 μL each of the primer solution from Step 1 into separate 0.2-mL siliconized PCR tubes for each sample and for a negative control."," :AP1r(1)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","from Step 1"," r(1)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","3. Add1μLoftotalRNA(i.e.,50ng)intothesampletubes.Add1μLofnuclease-freewaterintothe control tube. Mix the solutions several times for complete homogenization."," :AP1ai"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","If the total RNA is in a larger volume, reduce to 2 μL by centrifugal evaporation at room temperature (to avoid RNA degradation)."," a"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","The presence of sorbitol/trehalose will slow evaporation as the concentration increases, reducing the risk of accidentally drying the RNA."," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","99","4. Incubate the solutions at 65°C for 10 min in a thermocycler."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","5. Prepare 8 μL of the reverse transcription reaction mixture per reaction:"," :AP1im"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","per reaction:"," m"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","Reverse transcription reactions in the presence of betaine produce better nanoCAGE library outputs (Table 1)."," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","6. Pause the PCR machine at 22°C. Snap-cool the samples (from Step 4) in an ice-water bath for 2 min."," :AP1ir(4)x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","from Step 4"," r(4)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","RNAs must not refold,"," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","cooling occurs faster in an ice-water bath than on ice."," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","7. Return the samples to 22°C in the thermocycler."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","8. Add 8 μL of the reverse transcription reaction mixture (from Step 5) to the samples."," :AP1r(5)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","from Step 5"," r(5)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","9. Incubate for 10 min at 22°C, 30 min at 40°C, and 15 min at 75°C in the thermocycler."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","10. Immediately after finishing the reaction, snap-cool in an ice-water bath for 2 min."," :AP1x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","Checking the reverse transcription is not recommended at this step, because this will reduce the amount of cDNAs for PCR amplification and therefore increase the number of cycles needed."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","This removes smaller artifacts and primer dimers from the cDNAs that can make it difficult to determine the number of PCR cycles required for semisuppressive PCR."," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","Again, checking the purification at this step is not recommended."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","i. Use an Agencourt RNAClean XP kit according to the manufacturer’s instructions; mix the cDNA and beads with pipettes."," :AP1ir(x)x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","according to the manufacturer’s instructions"," r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","ii. Wash with 100 μL of 70% ethanol."," :AP1ix"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","Before eluting, remove as much ethanol as possible."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","Do not let the beads dry, because this can reduce recovery."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","iii. Elute with 40 μL of nuclease-free water."," :AP1ix"
"CSHP-2011-Salimullah-pdb.prot5559.txt","102","Quantitative real-time PCR is performed on a small scale to determine the number of cycles required for large-scale syn- thesis. This is necessary to keep the number of PCR cycles as low as possible (to reduce bias) and to suppress the syn- thesis of smaller artifacts."," :X1i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","12. For each sample, prepare 8.5 μL of real-time PCR mixture:"," :AP1mx"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","For each sample"," m"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","Perform real-time PCR in triplicate for the samples and control."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","13. Prepare one μltraAmp PCR plate. Add 8.5 μL of real-time PCR mixture per well."," :AP1m"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","per well"," m"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","14. Add 1.5 μL of purified cDNA or negative control (from Step 11.iii) to each well."," :AP1mr(11.iii)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","from Step 11.iii"," r(11.iii)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","to each well"," m"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","15. Seal the plate with adhesive PCR seal. Ensure a tight seal with a sealing applicator."," :AP1; AP1o"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","Centrifuge briefly if any solution has splashed above the mixture."," AP1o"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","if any solution has splashed above the mixture."," o"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","16. Perform quantitative real-time PCR using a StepOnePlus system according to the manufacturer’s instructions, with the following conditions:"," :AP1r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","according to the manufacturer’s instructions"," r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","i. Determine the “Cycle threshold: (CT)” values for each sample and control reaction after real-time PCR (Fig. 3)."," :AI1r(t)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","See Troubleshooting."," r(t)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","ii. Calculate the average CT values for each sample and for the negative control."," :AI2e"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","The average CT value for purified cDNA usually is ~16-20, depending on the source of RNA."," e"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","iii. Add 4 to the average CT value."," :AI1e"
"CSHP-2011-Salimullah-pdb.prot5559.txt","103","The optimum cycle number should be lower than 25 for the samples and very low or no signal for the negative control."," e"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","This procedure synthesizes the nanoCAGE cDNA libraries."," :X1i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","18. Prepare 85 μL of semisuppressive PCR mixture per reaction:"," :AP1ir(x)x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","Amplify purified samples in duplicate;"," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","The competition between primer annealing and self-annealing is the key mechanism of PCR suppression (Chenchik et al. 1998)."," r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","The concentration of the primers is thus lower than usual to strengthen the suppressive effect."," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","19. Add 85 μL of semisuppressive PCR mixture to each PCR tube."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","20. Add 15 μL of purified cDNA or negative control (from Step 11.iii) to each PCR tube."," :AP1mr(11.iii)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","from Step 11.iii"," r(11.iii)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","to each PCR tube"," m"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","21. Perform semisuppressive PCR using the following conditions:"," :AP1aer(17.iii)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","in Step 17.iii"," r(17.iii)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","is usually ~20-24 cycles"," e"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","PCR tubes can be left overnight in the PCR machine."," a"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","Perform purification and PCR at different locations and use different equipment to avoid cross-contamination."," :X1x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","i. For each sample, pool the PCR solutions from the duplicate tubes."," :AP1r(23.i)x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","Do not purify the negative control. It will be required for confirmation that the primer dimers and short PCR artifacts were eliminated from the amplified cDNAs"," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","see Step 23.i"," r(23.i)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","ii. Use an Agencourt AMPure XP kit according to the manufacturer’s instructions; mix the cDNA and beads with pipettes."," :AP1r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","according to the manufacturer’s instructions"," r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","iii. Wash with 600 μL of 70% ethanol."," :AP1x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","Before eluting, remove as much ethanol as possible. Do not let the beads dry, because this can reduce recovery."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","iv. Elute with 30 μL of nuclease-free water."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","i. Mix 2 μL of the purified nanoCAGE cDNAs with 3 μL of nuclease-free water and 1 μL of 6X loading dye. Mix 5 μL of nonpurified control (see Step 22.i.) with 1 μL of 6X loading dye."," :AP1r(22.i)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","see Step 22.i."," r(22.i)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","ii. Load the mixtures on a 1% agarose gel. Load 4 μL of a DNA marker."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","104","iii. Electrophorese until the markers separate in the range of from 100 bp to 2 kb (e.g., for agarose minigels, 15-30 min)."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","iv. Visualize the gel under UV light (see, e.g., Fig. 4)."," :AI1ir(t)v"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","Confirm their elimination by comparing the purified cDNAs with the nonpurified negative control."," v"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","Primer dimers or artifacts should not be present in the purified cDNAs."," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","See Troubleshooting."," r(t)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","24. Determine the concentration of the purified cDNAs in triplicate using a NanoDrop spectropho- tometer according to the manufacturer’s instructions. Calculate the average value."," :AP1r(x); AQ2er(Discussion)r(t)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","Calculate the average value."," AQ2"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","The concentration of the purified semisuppressive cDNA libraries should be >20 ng/μL to avoid artificial redun- dancy"," e"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","see Discussion"," r(Discussion)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","See Troubleshooting."," r(t)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","25. Dilute the samples with nuclease-free water to 10 ng/μL."," :AP2"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","This procedure adds adapter sequences to the nanoCAGE libraries for binding to the genome analyzer’s flow cell and for amplification by bridge PCR."," :X1i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","26. Prepare 92 μL of PCR mixture per reaction:"," :AP1ix"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","Prepare reactions in triplicate for each sample. Also prepare a control reaction without added template."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","The concentration of PCR primers is lower than usual because of the small number of cycles, which results in a large leftover."," i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","27. Add 92 μL PCR reaction mixture to each tube."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","28. Add8μL(i.e.,80ng)ofpurifiedDNA(fromStep25)toeachsampletube.Add8μLnuclease-free water to the control."," :AP1r(25)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","fromStep25"," r(25)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","29. Perform PCR using the following conditions:"," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","i. For each sample, pool the PCR solutions from the triplicate tubes."," :AP1x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","Do not purify the control."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","ii. Use an Agencourt AMPure XP kit according to the manufacturer’s instructions; mix the cDNA and beads with pipettes."," :AP1r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","105","according to the manufacturer’s instructions;"," r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","iii. Wash with 900 μL of 70% ethanol."," :AP1x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","Before eluting, remove as much ethanol as possible. Do not let the beads dry, because this can reduce recovery."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","iv. Elute with 30 μL of 0.1% Tween 20 in nuclease-free water."," :AP1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","31. Determine the concentration of the purified libraries in triplicate using a NanoDrop spectropho- tometer according to the manufacturer’s instructions. Calculate the average value."," :AP1r(x); AQ2e"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","Calculate the average value."," AQ2"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","The concentration of the purified library PCR products is usually >20 ng/μL (typically, ~20-30 ng/μL)."," e"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","It is important to check the removal of PCR primer dimers/artifacts, as well as to know the profile of the amplified libraries (e.g., size distribution, molar concentration) to calculate the amount of molecules to apply in the sequencing reaction."," :X1x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","32. Dilute 1 μL of each purified library to 5 ng/μL with 0.1% Tween 20."," :AP2"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","33. Analyze 1 μL each of the diluted samples using a High Sensitivity DNA Reagent Kit according to the manufacturer’s instructions."," :AP1r(t)r(x)x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","according to the manufacturer’s instructions."," r(x)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","Analyze the libraries in triplicate."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","See Troubleshooting."," r(t)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","This protocol assumes that only cDNAs in the 200-700-bp range are efficiently sequenced in the Gene AnalyzerIIx sequencer. There should be no peak shorter than 150 bp visible on the Bioanalyzer data."," x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","i. Open the library DNA analysis file (.xad)."," :AV1x"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","ii. Double-click on the electropherogram data of the sample to be analyzed."," :AV1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","iii. Click on the “Region table” icon under the page. Scroll into the table."," :AV1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","iv. Right-click the mouse button. Select “Modify region.”"," :AV1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","v. Input 200 and 700 in the boxes “From[bp]” and “To[bp]”, respectively."," :AV1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","106","vi. Click the “OK” button to determine the molarity."," :AV1"
"CSHP-2011-Salimullah-pdb.prot5559.txt","107","vii. Calculate the average concentration of the triplicate assays."," :AQ2"
"CSHP-2011-Salimullah-pdb.prot5559.txt","107","viii. Multiply this concentration with the dilution factor (from Step 32) to obtain the original library concentration."," :AQ1r(32)"
"CSHP-2011-Salimullah-pdb.prot5559.txt","107","35. Sequence the nanoCAGE libraries at a final concentration of 15 pM using an Illumina Genome AnalyzerIIx platform for single or paired-end reads."," :AV1i"
"CSHP-2011-Salimullah-pdb.prot5559.txt","107","One lane on this sequencer generally yields more than 15 million reads. Although not tested, a higher number of readings is possible with other systems (e.g., HiSeq2000 [Illumina])."," i"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","It can be performed a day in advance of chromatin extraction (or at any point if frozen chromatin aliquots are to be used)."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","This portion of the protocol requires 30 min followed by an overnight incubation. It can be performed a day in advance of chromatin extraction (or at any point if frozen chromatin aliquots are to be used)."," :X1ax"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","requires 30 min followed by an overnight incubation"," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","1. Resuspend magnetic Protein G (or Protein A) beads on a rotator for 5 min at roomt emperature."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","2. For each IP to be performed, aliquot 50 μL of beads into a microcentrifuge tube."," :AP1am"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","For each IP to be performed"," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","If several IPs will be performed using the same antibody, combine up to eight reactions worth of beads in a single 2-mL tube."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","3. Wash the beads with 1mL of bead binding buffer by resuspending the beads, placing the tubes on the magnetic stand for 1 min, and removing the bead binding buffer. Repeat."," :AP1a; CL(3)"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","Repeat."," CL(3)"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","If combining multiple aliquots of beads in a single tube, increase number of washes to five."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","4. Resuspend the beads in 200 μL of bead binding buffer per IP."," :AP1am"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","per IP"," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","If performing eight IPs, resuspend the beads in 1.6 mL of bead binding buffer in a 2-mL tube."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","5. Add up to 10 μg of the desired antibody per IP."," :AP1am"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","per IP."," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","The amount of antibody will vary considerably and will need to be empirically tested. Scale accordingly as above for multiple aliquots of beads."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","6. Incubate the beads and antibody on the rotator overnight at 4 ̊C."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","7. Wash the beads twice with 1 mL of bead-binding buffer and then twice with 1 mL of ChIP-Seq dilution buffer. Perform each wash by placing the tubes on the magnetic stand for 1 min and then removing the supernatant."," :AP1a"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","Increase number of washes to five bead-binding buffer washes and five ChIP-Seq dilution buffer washes if multiple reactions were combined above."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","8. Resuspend the beads in 50 μL of ChIP-Seq dilution buffer per IP to be performed."," :AP1m"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","per IP"," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","9. Store the beads at 4 ̊C until the chromatin is prepared."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","This procedure takes 1 h, not including any time necessary to harvest or dissect tissue."," :X1x"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","takes 1 h, not including any time necessary to harvest or dissect tissue."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","10. Transfer fresh or frozen tissue to a clean 1.5-mL tube containing 250 μL of ice-cold PBS."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","11. Briefly homogenize the tissue by hand to yield chunks 0.5 mm3 or smaller. Use a disposable plastic pestle or a handheld Polytron homogenizer fitted with 3.5-mm tip."," :AP1a"
"CSHP-2015-Cotney-pdb.prot084848.txt","3","Use a disposable plastic pestle or a handheld Polytron homogenizer fitted with 3.5-mm tip."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","12. Add ice-cold PBS to a final volume of 1 mL."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","13. Cross-link the tissue by adding 27 μL of 37% formaldehyde (1% final) to each tube and imme- diately invert the tube several times."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","14. Incubate the tube on the rotator at room temperature for 15 min."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","15. Quench the cross-linking by adding 67 μL of 2.5 M glycine (150 mM final) and incubate the tube on the rotator at room temperature for an additional 10 min."," :AP2"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","16. Harvest the tissue by centrifugation at 2000g for 10 min at 4 ̊C."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","17. Remove the supernatant and add 1 mL of ice-cold PBS. Flick the tube with your finger several times to dislodge the pellet."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","18. Harvest the tissue by centrifugation at 2000g for 10 min at 4 ̊C and discard the supernatant. Flash freeze the tissue pellet in liquid nitrogen and store at −80 ̊C."," :AP1; CB(19)o"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","Alternatively, proceed to Step 19 without freezing."," CB(19)o"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","This procedure requires 5 h."," :X1x"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","requires 5 h."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","19. Resuspend the frozen cross-linked tissue pellet in six volumes of ice-cold cell lysis buffer for ChIP-Seq. Use the markings on the 1.5-mL tube to provide an estimate of the pellet volume. If the pellet is very small, resuspend it in 300 μL of cell lysis buffer."," :AP1aci"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","Use the markings on the 1.5-mL tube to provide an estimate of the pellet volume."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","Cell pellets at this stage will frequently adhere to the insides of pipette tips. It may be necessary to resuspend the pellet by flicking the tube several times instead of by pipetting."," i"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","If the tissue remains in large chunks, homogenize it in a prechilled Dounce homogenizer with a loose pestle (pestle A) five to 10 times."," c"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","20. Incubate the tube on ice for 20 min."," :AP1i"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","This incubation period allows for swelling of cells to allow for mechanical lysis while keeping nuclei intact."," i"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","21. Transfer the tissue suspension to a prechilled Dounce homogenizer and homogenize with 30–40 strokes with the tight pestle (pestle B)."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","22. Transfer the homogenate to a fresh nonstick 1.5-mL tube, rinse the Dounce homogenizer with 500 μL of cell lysis buffer, and combine."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","23. Harvest the nuclei by centrifugation at 2000g for 5 min at 4 ̊C."," :AP1i"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","The pellet will be not be as tight against the tube as the original cell pellet and will be mostly white."," i"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","24. Remove the supernatant and resuspend the nuclei in five volumes of ice-cold ChIP-Seq nuclear lysis buffer (based on the volume of the original cell pellet in Step 19)."," :AP1ir(19)"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","in Step 19"," r(19)"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","The ChIP-Seq nuclear lysis buffer contains 0.2% SDS to aid in the lysis of nuclei. Some tissues such as brain samples require SDS concentrations of up to 0.5%; this will need to be empirically determined. High concentrations of SDS can inhibit antibody-binding downstream and will require further dilution or dialysis detailed below before using in IPs."," i"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","25. Incubate the tube on ice for 20 min."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","26. Take a 5-μL aliquot from each sample and dilute it with 15 μL of TE for an analytical gel."," :AP1ai"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","Store the aliquots on ice until required."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","This sample is to confirm that the chromatin was intact before isolation."," i"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","27. Place the tube in the sonicator bath set at 2 ̊C."," :AP1ax"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","In our hands, sonicating a volume of 300 μL per tube with the Misonix 431A cup horn has performed very consistently, so we advise dividing larger samples into multiple tubes for sonication."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","Place at least two and up to eight tubes in the tube holder in the sonicator."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","28. Adjust the water level so that the tubes just touch the surface of the cup horn."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","29. Sonicate the samples at amplitude 20 with 10-sec pulses and 10-sec rest for a total sonication time of 30 min."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","4","30. Remove the samples from the sonicator and store on ice."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","31. Take a 5-μL aliquot and dilute it with 15 μL of TE for an analytical gel."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","32. Treat all reserved aliquots with 10 μg of RNase A for 15 min at 37 ̊C."," :AP1m"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","all reserved aliquots"," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","33. Treat all reserved aliquots with 20 μg of proteinase K for 30 min at 65 ̊C."," :AP1m"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","all reserved aliquots"," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","34. Reverse the cross-links for 5 min at 95 ̊C and allow samples to cool slowly to room temperature."," :AP1x"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","These steps are required to ensure proper migration of the DNA on the agarose gel; otherwise, smearing or migration at much higher molecular masses than expected will occur."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","35. Add loading dye to each sample, and separate samples on a 1% agarose gel in 1× TAE running buffer."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","36. Visualize DNA samples on the agarose gel."," :AP1ar(t)v"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","Visualize"," v"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","heared chromatin should range in size from 200 to 500 bp."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","See Troubleshooting."," r(t)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","37. Centrifuge the samples from Step 30 at 16,000g for 10 min at 4 ̊C to remove insoluble material."," :AP1r(30)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","from Step 30"," r(30)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","38. Transfer the soluble sheared chromatin to fresh nonstick 1.5-mL tube. Take a final 5-μL aliquot and dilute it with 15 μL of TE for estimation of DNA concentration."," :AP1a"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","Chromatin can be frozen in liquid nitrogen and stored at −80 ̊C until needed."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","39. Treat the aliquot with RNase A, proteinase K, and cross-link reversal as above (Steps 32–34)."," :CL(32-34)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","40. Purify the chromatin aliquot with the QIAquick PCR Purification Kit, and measure the DNA concentration with PicoGreen and a NanoDrop 3300 Fluorospectrometer. Prepare a 1× Pico- Green stock and follow the manufacturer’s instructions for setting up a standard curve on the NanoDrop instrument."," :AP1r(x)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","follow the manufacturer’s instructions"," r(x)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","This procedure requires 10 min followed by an overnight incubation."," :X1x"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","requires 10 min followed by an overnight incubation."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","41. Dilute30–50μgofchromatinfromStep38withChIP-Seqdilutionbufferinafreshnonsticktube to reduce the SDS concentration <0.1% and achieve a final volume of 450 μL."," :AP1ar(38)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","from Step 38"," r(38)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","The amount of chromatin to be used will be highly antibody- and target-dependent and must be determined empirically, with 1–50 μg as a general range. Histones are highly abundant and widespread across the genome, whereas transcription factors are much less abundant and have specific binding sites."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","42. Reserve 5%–10% of diluted chromatin as an input sample and store at 4 ̊C."," :AP1i"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","Input must be reserved to assess enrichment via qPCR and to provide a necessary control for comparing sequence data to identify true binding sites or regions of enrichment."," i"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","43. Add 50 μL of the prepared antibody beads from Step 9 to each tube of diluted chromatin."," :AP1mr(9)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","to each tube of diluted chromatin."," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","44. Incubate tube on the rotator overnight at 4 ̊C."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","This procedure requires 2 h followed by an overnight incubation. Perform all washes and incubations in a cold room."," :X1x"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","requires 2 h followed by an overnight incubation."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","Perform all washes and incubations in a cold room."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","45. Remove the tube containing chromatin and the antibody beads from the rotator, place it on the magnet, and let it stand for 1 min."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","46. Remove the supernatant and transfer it to a fresh tube if required for additional IPs or troubleshooting."," :AP1o"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","if required"," o"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","47. Wash the beads from each IP with 1 mL of ChIP-Seq wash buffer by pipetting the beads up and down or inverting the tube several times to mix."," :AP1m"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","from each IP"," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","48. Place the tube on the rotator and incubate for 3 min."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","49. Place the tube on the magnet to precipitate the beads and remove supernatant."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","50. Repeat Steps 47 through 49 for a total of eight washes."," :CL(47-49)"
"CSHP-2015-Cotney-pdb.prot084848.txt","5","51. Perform one wash with 1 mL of TE. Remove the supernatant."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","52. Resuspend the beads in 85 μL of ChIP-Seq elution buffer."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","53. Elute the chromatin at 65 ̊C with agitation on the thermomixer for 10 min."," :AP1a"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","The beads will settle if not agitated frequently. If a thermomixer is not available, mix the beads by flicking the tubes with your finger every 2 min."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","54. Place the tube on the magnet and transfer the eluate to a fresh nonstick tube."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","55. Repeat elution with a second volume of 85 μL of ChIP-Seq elution buffer and combine eluates."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","56. Incubate the combined eluates at 65 ̊C overnight to reverse the cross-links."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","57. For each input control (see Step 42), add ChIP-Seq elution buffer to a final volume of 170μL and incubate overnight at 65 ̊C."," :AP2mr(42)"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","Foreachinputcontrol"," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","seeStep42"," r(42)"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","This procedure requires 4 h."," :X1x"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","requires 4 h"," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","58. Add 10 μg of RNase A to each tube of input and IP chromatin and incubate for 1 h at 37 ̊C."," :AP1m"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","to each tube of input and IP chromatin"," m"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","59. Add 200 μg proteinase K diluted in 120 μL TE to each tube and incubate for 2 h at 65 ̊C."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","60. Transfer each reaction to a 2-mL nonstick tube and add five volumes of PBI from the QIAquick PCR Purification Kit. Add 10 μL of 3 M sodium acetate to adjust the pH and generate a bright yellow color."," :AP1aw"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","Phenol:chloroform extraction and ethanol precipitation of DNA can be substituted here if preferred."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","However, depending on the amount of chromatin obtained, a significant volume of sample may be required for subsequent high-throughput sequencing library generation. Phenol carryover may inhibit initial end repair steps and result in low-quality libraries."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","FollowtheQIAquickPCRPurificationKitinstructionsandelutetheDNAwith50μLofEBintoa fresh nonstick 1.5-mL tube."," :AP1ar(x)"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","QIAquickPCRPurificationKitinstructions"," r(x)"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","EB can be prewarmed to 50 ̊C before elution to maximize recovery."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","62. Quantify the DNA recovered by IP and in the input sample with PicoGreen and a Fluorospectometer."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","63. Dilute a portion of the ChIP and input samples with dH2O to the same concentration in nonstick tubes (50–500 pg/μL)."," :AP2"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","64.4 Analyze target regions by qPCR using the following reaction:"," :AQ1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","i. Melt the template for 10 min at 95 ̊C."," :AP1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","ii. Amplify the target by performing 40 cycles of 15 sec at 95 ̊C and 1 min at 60 ̊C, measuring the fluorescence after each cycle."," :AP1; AQ1"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","iii. Examine the melting curve to ensure that a single product is made in each reaction."," :AP1r(x)"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","A good reference for performing qPCR is Bustin et al. (2009)."," r(x)"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","Measure enrichment by comparing Ct values between input and ChIP reactions."," :AQ1ae"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","Depending on the type of factor being immunoprecipitated, fold enrichments versus input can vary consider- ably."," a"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","For transcription factors and other sequence specific DNA-binding proteins, enrichments would be expected to be quite higher, up to 60-fold."," e"
"CSHP-2015-Cotney-pdb.prot084848.txt","6","For histone modifications, four- to 16-fold enrichments are acceptable."," e"
"CSHP-2015-Cotney-pdb.prot084848.txt","7","67. Dilute 10ng of ChIP or input DNA with dH2O to a final volume of 30μL in a fresh nonstick tube for high-throughput sequencing library preparation."," :AP2x"
"CSHP-2015-Cotney-pdb.prot084848.txt","7","Both ChIP and input samples must be sequenced to identify real binding peaks or enriched regions from more abundant open chromatin in the input sample."," x"
"CSHP-2015-Cotney-pdb.prot084848.txt","7","68. Follow the manufacturer’s instructions for preparing libraries with a kit."," :AP1ar(x)"
"CSHP-2015-Cotney-pdb.prot084848.txt","7","Follow the manufacturer’s instructions"," r(x)"
"CSHP-2015-Cotney-pdb.prot084848.txt","7","We routinely use kits from NEB (e.g., E6240) for sequencing on Illumina instruments. Many library prep kits for different sequencing instruments are now commercially available."," a"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","to each of the plates listed below."," m"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","1. Using a sterile toothpick, transfer cells of the yeast strain of interest to each of the plates listed below. Gently touch the toothpick containing cells to the center of each plate. On a separate set of plates, transfer cells of mutant strain defective for mat formation (a negative control; e.g., flo11Δ)."," :AP1imr(x)x"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","Gently touch the toothpick containing cells to the center of each plate."," x"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","0.3% agar is optimal to observe colony spreading. On these plates, colonies can show a radial spoke pattern."," i"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","2% agar is optimal to observe mat architecture. It also allows assessment of invasive growth by the plate- washing assay"," i"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","see The Plate-Washing Assay: A Simple Test for Filamentous Growth in Budding Yeast [Cullen 2015]"," r(x)"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","4% agar is optimal to observe colony ruffling and z-axis growth (see Fig. 1). The high surface rigidity reduces expansion in the plane of the xy-axis and promotes formation of dense architecturally complex mats that grow upward in the plane of the z-axis."," i"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","This plate maximizes complex colony morphology."," i"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","2. Maintain the plates in a 30 ̊C incubator in a location where vibrations are minimized. Examine mat expansion visually starting at 24 h and continuing over the course of several weeks."," :AP1ax"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","where vibrations are minimized"," x"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","Mats can be defined by their FLO11-dependent colony architecture and degree of expansion in the x-, y-, and z-axes by visual inspection (e.g., see Fig. 1)."," a"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","analyzing the photos with ImageJ software (http://imagej.nih.gov; Schneider et al. 2012)."," AV3"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","i. Measure the mat areas by photographing the plates and analyzing the photos with ImageJ software (http://imagej.nih.gov; Schneider et al. 2012)."," :AP1; AV3"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","ii. Examine the ruffled morphology characteristic of mats by photography with a digital camera at 1× to 5× magnification."," :AV3"
"CSHP-2015-Cullen-pdb.prot085076.txt","2","Reynolds et al. 2008"," r(x)"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","i. Place plastic wrap over the agar plate, and gently pull the plastic wrap off the plate."," :AP1ir(x)x"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","gently pull the plastic wrap off the plate"," x"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","When performing this action, the mat may or may not be removed from the agar. This distinguishes cells that adhere to the plastic from cells that adhere to the agar."," i"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","ii. Photograph both the plate and the wrap."," :AP1r(x)"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","iii. (Optional) Remove cells for additional analysis."," :AP1or(x)"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","Optional"," o"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","i. Atvarioustimesaftermatformationhasoccurred,removecellsusingatoothpick.Resuspend in 500 μL of H2O."," :AP1x"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","Always compare the cells of interest with flo11Δ cells (a negative control)."," x"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","ii. Adjust the optical density of the cells to A600 = 2.0."," :AV1"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","iii. Add 100 μL of the cell suspension to a 96-well polystyrene or polypropylene plate."," :AP1w"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","96-well polystyrene or polypropylene plate"," w"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","iv. Incubate the cells for 4 h at 25 ̊C to allow the cells to settle to the bottom of the wells."," :AP1"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","v. Add 100 μL of 1% crystal violet to each well. Incubate for 20 min at 25 ̊C."," :AP1"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","vi. Wash wells five times with water and photograph adherent cells with a digital camera or by microscopy at 10×."," :AP1"
"CSHP-2015-Cullen-pdb.prot085076.txt","3","Wild-type cells will adhere to the plastic surface and will be violet in color. In contrast, flo11Δ cells will not adhere, leaving a transparent plastic surface that is relatively free of cells."," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","1. Collect the embryos in PBS or tissue culture medium."," :AP1a"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","If the embryos are at the prestreak to early somite stages, it is advisable to add some serum (~5%) to reduce stickiness (this will cause a protein precipitate in the fixative, but do not be concerned)."," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","2. Dissect away the extraembryonic membranes to facilitate penetration of the antibodies."," :AP1i"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","to facilitate penetration of the antibodies."," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","i. For early somite-stage mouse embryos (up to ~eight somites), use the tips of fine forceps to flatten the embryo by pushing the edges of the visceral yolk sac into the surface of a plastic Petri dish."," :AP1io"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","For early somite-stage mouse embryos"," o"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","By “pinning out” the embryos in this way, they can be fixed flat and will remain extended after being detached from the dish. The extended shape allows better viewing of the somites and neural tube after staining."," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","ii. For older embryos (9.5-10.5 d), it is sometimes best to cut them in half sagitally before or after fixation."," :AP1aio"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","For older embryos"," o"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","it is sometimes best to"," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","This makes it easier to visualize and photograph staining of bilaterally symmetrical structures such as the cranial nerves, which otherwise appear as “double images.”"," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","3. Fix the embryos in freshly prepared methanol/DMSO (4:1) at 4°C overnight."," :AP1ir(x)"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","DMSO permeabilizes the tissues"," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","See CSH Protocols article Handling Mouse Blastocysts for Fixation."," r(x)"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","4. Transfer the embryos into freshly prepared methanol/DMSO/H2O2 (4:1:1) at room temperature for 5-10 h."," :AP1i"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","This blocks endogenous peroxidase activity (e.g., in red blood cells)."," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","5. Store the embryos (individually or in small groups) in 100% methanol at -20°C."," :AP1a"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","individually or in small groups"," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","2","The embryos may be stored for at least several months and probably longer."," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","i. Add 1 mL of 50% methanol and incubate for 30 min with rocking. Remove the solution with a Pasteur pipette or micropipette."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","ii. Add 1 mL of PBS and incubate for 30 min with rocking. Remove the PBS."," :AP1c"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","If embryos stick to the sides, siliconize the tubes."," c"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","iii. Add 1 mL of PBSMT and incubate for 1 h with rocking (do this twice, removing the solu- tion after each wash)."," :AP1ix"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","Rocking is important to facilitate penetration of the antibody: Ensure that the solution is mixing well."," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","However, the embryos are fragile once they are fixed, so do not rock them too hard."," x"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","7. Add 1 mL of primary antibody diluted in PBSMT. Incubate the embryos overnight at 4°C with rocking."," :AP1ai"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","If the antibody is stable, this and all subsequent procedures can be performed at room temperature."," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","The correct dilution must be determined empirically, but 1:200 is a typical dilution."," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","i. Wash once in 1 mL for 1 h at 4°C. Transfer the embryos to a 15-mL tube."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","ii. Wash once with 10 mL for 1 h at 4°C."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","iii. Wash three times in 10 mL for 1 h at room temperature."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","9. Transfer the embryos to microcentrifuge tubes. Add 1 mL of secondary antibody diluted 1:500 in PBSMT, and incubate them overnight at 4°C with rocking."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","10. Wash the embryos again as in Step 8."," :AP1r(8)"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","11. Rinse the embryos in 5 mL of PBTX, and then transfer them to microcentrifuge tubes. Wash them in 1 mL of PBTX with rocking for 20 min."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","12. Incubate the embryos in microcentrifuge tubes with 1 mL of DAB-NiCl2 at room temperature for 30 min."," :AP1i"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","This allows full penetration of the substrate into the embryo."," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","13. Add H2O2 to a final concentration of 0.03% and rock until the color intensity in the embryos looks good (i.e., specific regions of staining are obvious and before background staining comes up; usu- ally 2-10 min)."," :AP2a"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","If necessary, check color in the dissecting microscope. If the color reaction occurs too quickly, use a lower con- centration of H2O2."," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","14. Post-fix the embryos in 4% paraformaldehyde."," :AP1i"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","Without post-fixation, the color of the stained embryos will fade under strong light, particularly under light used for photography."," i"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","i. Quickly rinse in 1 mL of PBTX."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","ii. Washin1mLofPBTXfor30min."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","iii. Wash in 1 mL of 50% methanol for 30 min."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","iv. Wash in 1 mL of 80% methanol for 30 min."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","v. Wash in 1 mL of 100% methanol for 30 min."," :AP1a, CB(17)1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","If necessary, whole-mount embryos can be sectioned."," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","If this is the case, proceed to Step 17."," CB(17)1"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","16. Remove the methanol, add 500 μL of BABB, and rock for 10 min. Make observations of the stained embryos in a glass Petri dish (do not use polystyrene dishes). For photography, place embryos in a depression slide with a coverslip on top. Use a tungsten color film without filters or a daylight color film with a blue filter."," :AP1avx"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","Make observations of the stained embryos in a glass Petri dish (do not use polystyrene dishes)"," v"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","For photography, place embryos in a depression slide with a coverslip on top."," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","do not use polystyrene dishes"," x"
"CSHP-2008-Joyner-pdb.prot4820.txt","3","Use a tungsten color film without filters or a daylight color film with a blue filter."," x"
"CSHP-2008-Joyner-pdb.prot4820.txt","4","Although embryos can be stored in BABB in a dark place, it is advisable to obtain a photographic record as soon as possible."," a"
"CSHP-2008-Joyner-pdb.prot4820.txt","4","i. Wash twice in 100% ethanol for 1 h each."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","4","ii. Wash twice in xylene for 1 h."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","4","iii. Wash once in xylene:wax (1:1) for 1 h."," :AP1"
"CSHP-2008-Joyner-pdb.prot4820.txt","4","iv. Wash twice in wax for 1 h each."," :AP1, CL(17.iv)1"
"CSHP-2008-Joyner-pdb.prot4820.txt","4","v. Embed, section, and mount the embryos (for details, see CSH Protocols articles Embedding Mouse Embryos and Tissues in Wax and Sectioning Mouse Embryos). For counterstain- ing, use a light (30-45 sec) stain with eosin B."," :AP1ar(x)"
"CSHP-2008-Joyner-pdb.prot4820.txt","4","for details, see CSH Protocols articles Embedding Mouse Embryos and Tissues in Wax and Sectioning Mouse Embryos"," r(x)"
"CSHP-2008-Joyner-pdb.prot4820.txt","4","For counterstain- ing, use a light (30-45 sec) stain with eosin B."," a"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","i. Set heat at 12.5-13.0 and set the solenoid at 4 on the needle puller."," :AP1w"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","ii. Follow the manufacturer’s instructions for pulling the needles."," :AP1r(x)"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","iii. Store freshly-pulled needles horizontally, embedded lightly on a flattened narrow strip of modeling clay placed on the bottom of a 150-mm covered plastic Petri dish."," :AP1"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","2. Use a pipette (e.g., Eppendorf P10) fitted with a microtip (e.g., Eppendorf Microloader tips) to back-load the injection needles with 0.5-1.0 μL of dsRNA mixed with filtered food dye."," :AP1w"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","3. Set the Transjector to automatic injection."," :AP1"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","4. Set the injection pressure (Po) to 450."," :AP1"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","5. Set the compensation pressure (Pc) to 570."," :AP1"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","6. Set the time of injections to 0.1 sec."," :AP1r(x)"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","If using a different model injector, calibrate the injector according to the manufacturer’s instructions."," r(x)"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","Based on the volume loaded into the needle (0.5-1.0 μL), each embryo receives up to 100-200 pL of dsRNA. A typical load- ing, if the needle remains unbroken and unblocked, is good for the injection of approximately 1000 embryos or more."," :X1i"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","Inject the embryos in the posterior end, slightly off-center, because the posterior tip of the chorion is very hard and needles often break in this position. Keep the injection room at 18°C to slow development."," :X1ax"
"CSHP-2008-Misquitta-pdb.prot4918.txt","2","i. Move the slide slowly toward the needle tip while simultaneously depressing the “clean” button on the Transjector."," :AP1"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","ii. The moment that the slide lightly taps the tip and the tip breaks, a small amount of the dye will leak from the needle tip. Release the “clean” button."," :AP1ex"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","Dye flow should stop when the “clean” button is no longer depressed."," e"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","If the tip is too large after breakage, the needle will continue to drip dye when the “clean” button is not depressed. Such tips will destroy the embryos during injection."," x"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","8. Bring the needle tip and the posterior tip of the embryo into the same focal plane. Make sure that the needle is positioned off-center with respect to the posterior tip of the embryo (see Fig. 1)."," :AP1ax"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","Make sure that the needle is positioned off-center with respect to the posterior tip of the embryo (see Fig. 1)."," x"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","For best results, inject the embryos with the needle at an angle slightly greater than 45° relative to the embryo surface in the posterior quarter of the embryo."," a"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","9. Insert the needle just far enough to penetrate the vitelline membrane."," :AP1x"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","just far enough to penetrate the vitelline membrane"," x"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","10. Inject the RNA into the embryo. A small amount of food dye will appear as a small dot in the posterior end of the embryo. It takes ~1-5 sec to inject each embryo."," :AP1x"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","It takes ~1-5 sec to inject each embryo."," x"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","11. After all the embryos on a slide have been injected, place the slide in a covered moist chamber at 18oC-22oC until embryogenesis is complete (~48 h) or until the desired stage of development is reached."," :AP1"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","12. Wash the embryos off the slide into a nylon-mesh collection basket."," :AP1"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","13. Transfer the embryos to a microcentrifuge tube."," :AP1"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","14. Fix the embryos and stain them using standard protocols; see Ashburner (1989a,b)."," :AP1r(x)"
"CSHP-2008-Misquitta-pdb.prot4918.txt","3","standard protocols; see Ashburner (1989a,b)"," r(x)"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","1. Trypsinize cells that have been growing in tissue culture dishes with or without pretreatment with a mitoic arrestant. Pipette to give a single cell suspension. Add medium and serum and centrifuge at 1000 rpm in a 2-mL conical glass tube for 5 min."," :AP1r(x)"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","For guidance in trypsinization, see Trypsinization of Cells Grown in Monolayer (Darlington 2006)."," r(x)"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","2. Resuspend the cell pellet gently in 1 mL of aqueous 0.56% (w/v) KCl by adding drops and flick- ing the tube. Do not pipette up and down. Add more KCl to a final volume of 4 mL. Leave at room temperature (21°C-23°C) for 6 min to swell the cells."," :AP1x"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","Do not pipette up and down."," x"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","3. Centrifuge the cells again at 1000 rpm for 5 min. Carefully remove the supernatant, and fix the cell pellet in 1 mL of fresh Carnoy’s fixative at room temperature. Resuspend by flicking the tube. Make up to 4 mL. Then centrifuge to pellet the cells. Repeat three times."," :AP1, CL(3)1"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","4. Centrifuge the cells at 1000 rpm for 5 min and then resuspend them in a small volume (0.5 mL) of fixative."," :AP1ar(5)"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","Slide preparations (see below) are best made within 0-3 h of the final fixation of the cells, but they can be made up to 7 d afterward if the material is kept at 4°C and the cell pellet is resuspended in fresh fixative before attempting to spread the cells. The longer the material is kept, the poorer the spreading quality of the mitotic cells. If possible, at least five slides should be made from each sample to guarantee success."," a"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","see below"," r(5)"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","5. Clean commercially purchased, precleaned slides further by leaving them overnight in a mixture of absolute ethanol and concentrated HCl (1:1). The next day, wash the slides in running tap water, rinse in deionized H2O, and store in absolute ethanol and diethyl ether (1:1)."," :AP1"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","6. Shortly before using the slides, use forceps to remove slides from the mixture of absolute alcohol and diethyl either. Wipe the slides dry on Kimwipes or some other coarse, grease-free tissues, avoiding finger contact with the slide to prevent making fingerprints on the slide surfaces."," :AP1x"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","avoiding finger contact with the slide to prevent making fingerprints on the slide surfaces"," x"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","7. To make chromosome spreads on these slides, add a row of three drops of cell suspension from a prepared Pasteur pipette (~0.2-mL capacity, drawn to deliver 10-μL drops), allowing them to spread to their maximum size. Let the drops dry until interference rings are visible at their periph- ery. Drying can be assisted by blowing on the slide while holding it up to the heat generated by a desk lamp bulb."," :AP1a"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","Drying can be assisted by blowing on the slide while holding it up to the heat generated by a desk lamp bulb."," a"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","8. Once dry, inspect the slides under low-power phase-contrast microscopy (final magnification 160X). If the density of cells and chromosome spreads is insufficient, pellet the cells, resuspend in a smaller volume of fixative, and repeat with fresh drops."," :AP2cv"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","inspect the slides under low-power phase-contrast microscopy"," v"
"CSHP-2008-Nagy-pdb.prot4706.txt","2","If the density of cells and chromosome spreads is insufficient, pellet the cells, resuspend in a smaller volume of fixative, and repeat with fresh drops."," c"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","9. For counting chromosomes without banding, stain in Giemsa for ~15 min and photograph with green filter."," :AP1"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","0. Place suitably aged slides (see note below) in 2X SSC in a Coplin staining jar with a lid in a water bath at 60°C-65°C for 1.5 h. Then cool the slides to room temperature by running tap water over the closed jar. Transfer the slides to 0.85% (w/v) NaCl at room temperature for 5 min."," :AP1aix"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","Before G banding, slides should be “aged” for between 3 and 21 d by leaving them in a closed box at room tem- perature. Fresh slides give poor G-band resolution."," a"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","Maximum G-band resolution is achieved at ~10 d after slide preparation."," i"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","Beyond this time, resolution slowly decreases until, after several weeks in storage, the chromosomes either fail to band and stain uniformly or show “pseudobands” that are not significant to the standard idiogram."," x"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","11. Drain the slides by touching them onto filter paper. Place them on a flat surface and flood the chamber with 0.025% trypsin in 0.85% NaCl for 15-20 sec."," :AP1air(x)"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","The trypsin exposure time is critical: Underexposure preserves chromosome morphology but gives poorly differ- entiated bands, and overexposure distorts morphology and eliminates most of the bands. Optimum trypsin times are known to vary among laboratories."," i"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","A test slide should be treated for the minimal suggested time of 15 sec to establish the best treatment time for the rest of the slides."," a"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","In the laboratory of E.P. Evans, the optimal trypsin exposure time has been established as between 15 and 20 sec for mouse chromosomes, irrespective of the source of the mitotic cells."," r(x)"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","12. Stop tryptic activity by placing the slides back into 0.85% NaCl. Then rinse slides in phosphate buffer (pH 6.8) and stain in fresh Giemsa stain in 5 mM phosphate buffer (pH 6.8). After 10 min in the stain, monitor wet slides under low-power, bright-field microscopy (160X) for staining inten- sity. Because, upon drying, wet slides gain contrast, care should be taken not to overstain the cells as this will reduce G-band differentiation. If necessary, repeat staining until adequate results are achieved and then quickly rinse slides in phosphate buffer (pH 6.8) and blow-dry with a current of cool air."," :AP1x; W3; CL(12)1o"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","After 10 min in the stain, monitor wet slides under low-power, bright-field microscopy (160X) for staining inten- sity."," W1"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","Because, upon drying, wet slides gain contrast, care should be taken not to overstain the cells as this will reduce G-band differentiation."," x"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","If necessary, repeat staining until adequate results are achieved and then quickly rinse slides in phosphate buffer (pH 6.8) and blow-dry with a current of cool air."," CL(12)1o"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","13. Examine unmounted slides with a 100X oil-immersion lens."," :AV3aiv"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","The majority of modern, readily available immersion oils that are declared “PCB-free” also have the unfortunate property of removing Giemsa stain after a few hours of exposure."," i"
"CSHP-2008-Nagy-pdb.prot4706.txt","3","Although direct viewing of slides under an oil immersion lens gives a higher optical resolution, it is wise to mount slides if they are to be kept."," a"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","1. Sterilize Arabidopsis seeds by rinsing with 75% ethanol for 5 min, followed by a 1-min rinse in 95% ethanol."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","2. Dry the seeds on autoclaved filter paper in a laminar flow hood."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","3. Plate the sterilized seeds on 0.5X MS medium. Stratify in darkness for 2 d at 4°C."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","4. Transfer the plates to a growth chamber. Grow for 7-10 d using an 8/16 h light/dark cycle (130 μmol/m2 sec light intensity), 21°C ± 1°C light temperature/18°C ± 1°C dark temperature and 70%- 80% humidity."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","5. After 7-10 d, transplant the seedlings into pots filled with soil mix. Continue to grow under the conditions indicated in Step 4."," :AP1r(4)"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","6. Harvest 5-10 fully expanded young leaves from 4-wk old Arabidopsis plants (Fig. 1)."," :AP1vw"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","Harvest 5-10"," w"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","Fig. 1)"," v"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","7. Using sharp-tip forceps, peel the abaxial epidermis from the leaves."," :AP1ix"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","Using sharp-tip forceps,"," x"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","This decreases mesophyll contamination."," i"
"CSHP-2008-Zhang-pdb.prot5014.txt","2","8. Place the epidermal peels in a blender with 500 mL of cool distilled water. Blend for 30 sec."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","3","9. Filter the mixture through a 100-μm nylon mesh. Using a spatula, transfer the peels from the mesh into 10 mL of enzyme solution 1."," :AP1v"
"CSHP-2008-Zhang-pdb.prot5014.txt","3","Peels with guard cells after blending are shown in Fig. 2A."," v"
"CSHP-2008-Zhang-pdb.prot5014.txt","3","10. Digest the peels in a shaking water bath at 120 rpm for 30 min at 28°C."," :AP1v"
"CSHP-2008-Zhang-pdb.prot5014.txt","3","Peels at this stage are shown in Fig. 2B)."," v"
"CSHP-2008-Zhang-pdb.prot5014.txt","3","11. Add 10 mL of basic solution. Shake for an additional 5 min."," :AP1i"
"CSHP-2008-Zhang-pdb.prot5014.txt","3","This step reduces the osmolality difference between enzyme solutions 1 and 2."," i"
"CSHP-2008-Zhang-pdb.prot5014.txt","3","12. Collect the partially digested peels by filtering the solution through a 100-μm nylon mesh. Using a squirt bottle, wash the peels with basic solution. Transfer the peels to 10 mL of enzyme solution 2."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","3","13. Digest the peels by shaking at 60 rpm for at least 30 min at 22°C."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","i. After 30 min, remove an aliquot of peels at 5-min intervals."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","ii. Using a microscope (100X and 400X total magnification), evaluate the degree of digestion."," :AQ3v"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","Peels at different digestion stages are shown in Fig. 2C-E)."," v"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","iii. Stop the digestion when 10%-20% of the guard cells are spherical (Fig. 2D)."," :AP3v"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","14. Mix the peels by pipetting up and down with a 1-mL pipette."," :AP1i"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","This releases the protoplasts from the peels."," i"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","15. Filter the solution containing the protoplasts through a 30-μm nylon mesh to remove debris. Using a squirt bottle, wash the mesh with ~5 mL of basic solution. Collect both the filtrate and the wash solution into a centrifuge tube."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","16. Centrifuge the solution containing the protoplasts at 800 rpm for 5 min to pellet the protoplasts. Decant the supernatant."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","17. Gently resuspend the pellet in 15 mL of basic solution. Repeat Step 16."," :AP1; CL(16)1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","18. Gently resuspend the pellet in 200 μL of basic solution. Place on ice in the dark for at least 1 h prior to patch clamping."," :AP1r(t)"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","See Troubleshooting."," r(t)"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","Patch clamping should always be performed with pipettes prepared on the same day."," :X1x"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","19. Pull each glass capillary tube into two pipettes using a two-step puller. Set the temperature for each step properly to make sure the tip of the pipette is sharp and thin, with a tip opening of <1 μm."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","20. Smooth the sharp tip of the pipette by polishing the tip with a microforge. Stop heating the tip when the tip opening becomes very small."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","21. Store the polished pipettes mounted on a clay strip in a covered dish to avoid contamination and clogging of the pipette tip."," :AP1i"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","The seal resistance of the pipette should be 40-60 MΩ using the described patch clamping solutions."," i"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","22. Fill the cell chamber with bath solution. Add 20 μL of the protoplast solution (from Step 18). Wait 5 min to allow the protoplasts to settle to the bottom of the chamber (Fig. 2F)."," :AP1r(18)v"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","from Step 18"," r(18)"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","Fig. 2F"," v"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","23. Place the tip of the ground electrode into the bath solution via a 3 M KCl agar bridge."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","24. Turn on the amplifier and computer. Load pCLAMP software (Clampex) for whole cell data acquisition. Load or edit the proper parameters to set the voltage family to evaluate guard cell K+ channels:"," :AP1ivx"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","i. A typical voltage family for inward and outward K+ channel current recordings consists of negative and positive voltages (e.g., -200 mV to +80 mV with 20-mV increments)."," i"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","See the waveform example in Fig. 3."," v"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","ii. Set the duration to no less than 4 sec for each voltage step to make sure the currents reach steady state, with a holding potential of -60 mV and an interpulse interval of 6 sec."," x"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","25. Heat the tip of the plastic syringe using an alcohol burner. Pull the syringe into a very thin tube with a diameter <0.5 mm."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","26. Using the syringe, fill a glass pipette with pipette solution. Remove air bubbles by flicking the pipette."," :AP1i"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","Air bubbles can cause an open circuit, preventing recording of the current."," i"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","27. Tighten the pipette into the holder, making sure that only 1-2 mm of the Ag/AgCl electrode wire is submerged in the pipette solution."," :AP1x"
"CSHP-2008-Zhang-pdb.prot5014.txt","4","making sure that only 1-2 mm of the Ag/AgCl electrode wire is submerged in the pipette solution"," x"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","28. Using the inverted microscope, select a round, bright, healthy protoplast with a clear membrane (as indicated by the arrow in Fig. 2F). Move it to the center of the field of view."," :AV1v"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","as indicated by the arrow in Fig. 2F"," v"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","29. Use the coarse manipulator to move the glass pipette so that its tip is centered over the protoplast and above the surface of the bath solution."," :AP1"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","30. Reduce the light intensity of the microscope so that it is easier to find the shadow of the pipette tip. While observing using a 10X objective, use the coarse manipulator to move the pipette down until the tip is above and very close to the selected protoplast."," :AP1x"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","using a 10X objective"," x"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","To avoid clogging the pipette tip, maintain positive pressure by blowing into the tube that is connected to the electrode holder."," x"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","31. While maintaining positive pressure, change to the 40X objective. Use the fine micromanipulator to move the pipette so that it gently touches the protoplast, very slightly deforming it."," :AP1x"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","very slightly deforming it"," x"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","32. Apply gentle suction to the tube so that the tip of the glass pipette seals onto the protoplast."," :AP1r(t)"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","See Troubleshooting."," r(t)"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","33. Continue to apply suction. Set the pipette offset to zero. Monitor the increase in the seal resist- ance on the computer. Gradually apply a more negative holding potential (-10 mV for each 100 MΩ increase in seal resistance) to -60 mV."," :AP1r(t)"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","See Troubleshooting"," r(t)"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","34. When a GΩ seal resistance is reached, give a stronger pulse of suction and/or a voltage zap from the patch clamp amplifier."," :AP1ir(t)"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","This breaks the membrane and forms the whole-cell configuration."," i"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","See Troubleshooting"," r(t)"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","35. If the seal resistance remains 1-2 GΩ or higher, apply test voltages to the cell. View whole-cell K+ currents and record for further analysis (Fig. 3)."," :AP1; AV3av"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","Fig. 3"," v"
"CSHP-2008-Zhang-pdb.prot5014.txt","5","and 5 GΩ can also be used if leak (instantaneous) currents are not too large. For recording of single channel currents from membrane patches, seal resistances of at least 10 GΩ are required in order to achieve a suitable signal-to-noise ratio."," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","3","Expression of cell-membrane-targeted fluorescent proteins (e.g., memGFP) provides an excellent means for examining cell morphology in living tissues."," i"
"CSHP-2010-Kieserman-pdb.prot5427.txt","3","i. Inject 60-500 pg of mRNA for visualization at gastrula stages."," :AP1i"
"CSHP-2010-Kieserman-pdb.prot5427.txt","3","Because overexpression can lead to ectopic or abnormal protein localization, care should be taken to inject the lowest dose of mRNA that allows visualization of the construct. A careful dose-curve experiment should be performed to determine the proper expression level empirically before proceeding. Injections of 15-75 pg of mRNA are typical, but concentrations as low as 5 pg or as high as 500 pg might be needed."," ax"
"CSHP-2010-Kieserman-pdb.prot5427.txt","3","ii. Inject the mRNA of interest."," :AP1ar(3)x"
"CSHP-2010-Kieserman-pdb.prot5427.txt","3","When imaging (see Step 3), use a high-NA objective."," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","3","see Step 3"," r(3)"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","iii. Inject 4-cell embryos with an mRNA encoding a fusion protein of interest."," :AP1ior(x)v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Alternatively, mosaic expression can also be achieved by injection of plasmid DNA"," o"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","(Vize et al. 1991)"," r(x)"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Because plasmid DNA is not transcribed until after the mid-blastula transition, this approach avoids early expression of the marker gene, which might be desirable. Moreover, expression levels will vary from cell to cell using this approach"," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Fig. 5C"," v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","iv. Inject again at a later cleavage stage to manipulate gene function in only a subset of cells expressing the reporter. In this later injection, include both the morpholino-oligonucleotide or mRNAs to manipulate gene function and also a complementary lineage tracer to mark the manipulated cells."," :AP1aivx"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","In this later injection, include both the morpholino-oligonucleotide or mRNAs to manipulate gene function and also a complementary lineage tracer to mark the manipulated cells."," x"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Large clones of manipulated cells can be made with second injections at the eight-cell stage"," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Fig. 6A"," v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Smaller clones of manipulated cells can be made with second injections at the 16-cell to 32-cell stages"," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Fig. 6B"," v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Plasmid DNA can also be injected following mRNA injection, allowing mosaic expression of one marker in a uniform background of a complementary marker"," i"
"CSHP-2010-Kieserman-pdb.prot5427.txt","4","Fig. 5"," v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","These methods for mounting and imaging are also effective for fixed embryos following immunostaining or in situ hybridization, provided that clearing is not required. For imaging cleared embryos, see Preparation of Fixed Xenopus Embryos for Confocal Imaging (Wallingford 2010b)."," ir(x)"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","See Figure 7A for an illustration of immobilibzation at these stages."," v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","i. Pour a layer of 2% agarose into the imaging chamber."," :AP1ir(x)v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","ii. Using a small comb, make embryo-sized wells in the agarose."," :AP1"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","iii. Devitellinize embryos. Place them into the wells such that the surface of interest can be imaged (e.g., the neural plate during neural tube closure [Lee et al. 2007; Kieserman et al. 2008]; the animal cap of blastula stages in whole mounts [Woolner et al. 2008])."," :AP1r(x)x"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","e.g., the neural plate during neural tube closure [Lee et al. 2007; Kieserman et al. 2008]"," r(x)"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","Take care to position the embryo as close as possible to the cover glass bottom to accommodate the working distance of the objective. Also, the less agarose between sample and objective, the better. This method is effective for imaging up to 6 h."," x"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","the animal cap of blastula stages in whole mounts [Woolner et al. 2008]"," r(x)"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","The epidermis of tailbud and tadpole stage Xenopus embryos is an excellent in vivo model for studying epithelial biology (Hayes et al. 2007; Kieserman et al. 2008; Park et al. 2008). Imaging this tissue also uses the specialized chamber, but involves a different mounting approach (Fig. 7B). This approach is also effective for long-term time-lapse analysis (>12 h)."," air(x)v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","iv. Place the embryos on the cover glass of an imaging chamber in a small drop of 1/3X MMR."," :AP1air(x)v"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","v. Working quickly, remove the MMR entirely. Replace with a drop of lukewarm 0.8% low- melt agarose. Use a hair loop to quickly position the embryo as desired."," :AP1x"
"CSHP-2010-Kieserman-pdb.prot5427.txt","5","Working quickly"," x"
"CSHP-2010-Kieserman-pdb.prot5427.txt","6","vi. After the agarose hardens, fill the chamber with 1/3X MMR to prevent desiccation."," :AP1i"
"CSHP-2010-Kieserman-pdb.prot5427.txt","6","The sample is now ready for imaging."," i"
"CSHP-2010-Kieserman-pdb.prot5427.txt","6","When setting up the imaging parameters, a critical consideration is the balance between photobleaching and the brightness of the sample. To err on the side of caution with regard to photobleaching, decrease the laser power and then compensate by increasing the detector gain or increase the brightness post-acquisition; care must be taken with post-acquisition processing (see Step 6). At high magnification, one can reliably image to a depth of only about one cell diameter. Higher laser power can add depth, but is clearly phototoxic. In some cases (usually at lower magnifications), bright signals underneath unlabeled cells can be imaged clearly. For example, the crawling myeloid cells in Figure 2 are labeled and imaged through two to three layers of unlabeled epidermal cells. Imaging deeper tissues can almost always be achieved by imaging the cells in organotypic culture (see Discussion)."," ar(6)vx"
"CSHP-2010-Kieserman-pdb.prot5427.txt","6","i. Collect stacks of slices over time (Fig. 8)."," :AV3ar(6)r(x)vx"
"CSHP-2010-Kieserman-pdb.prot5427.txt","6","The size of stacks, the amount of overlap between the slices within stacks (z-resolution), and the collection times (time-resolution) must be optimized empirically for your specimen, fluorophore, and application"," x"
"CSHP-2010-Kieserman-pdb.prot5427.txt","6","see, e.g., Kieserman et al. 2008, Supplemental Table 2"," r(x)"
"CSHP-2010-Kieserman-pdb.prot5427.txt","6","ii. Image with 40X, 63X, or 100X objectives."," :AV1w"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","iii. Image with 5X-20X objectives."," :AV1aw"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","Imaging at 40X is also possible, although the lower magnifications are preferred."," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","4. Collect images as a stack of individual tiff files for a time-lapse movie."," :AV1"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","5. Check the movie every 15-30 min during acquisition. Correct focus drift or embryo movement as needed."," :W3a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","For long-term imaging of tadpole stages, add a solution of 0.15% Tricaine to the medium to prevent twitching of the embryo."," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","6. With careful imaging, little or no post-acquisition processing is needed. If necessary, however, perform image processing to improve the image for the computer screen and printed page:"," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","Many journals now request that all original, unprocessed images be deposited with the journal. The original files MUST therefore be saved. ALWAYS create a renamed copy of images before any processing is performed (e.g., by adding “_PROCx” to the original file, where x is a number; different iterations of the processing thus get consecutively higher numbers) and perform enhancements on the renamed copy. Always apply filters and enhancements to the entire image. NEVER apply enhancements or filters to selected regions of an image. Like imaging, post-processing is most effective if trial and error is used."," x"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","i. Contrast can be enhanced using Photoshop’s “Levels” function (Image Adjustments Levels)."," :AV1aix"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","This function is more useful than the Brightness/Contrast function."," i"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","ii. Image clarity can be improved using Photoshop’s “Unsharp Mask” function (Filter Sharpen Unsharp Mask)."," :AV1"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","iii. Noise can be reduced using the Median (Filter Noise Median) or Dust & Scratches (Filter Noise Dust & Scratches) filter."," :AV1"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","7. Use Photoshop to resize (Image Image Size) and compress (File Save As) individual tiff files."," :AV1ar(9)w"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","This can be done for all the images using the File Automate Batch function."," a"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","lternatively, the movie can be resized after assembly (see Step 9). Tiny movies are useful for e-mailing to collaborators, but the largest, highest quality movies that are practical should be submitted with a paper for review."," w"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","see Step 9"," r(9)"
"CSHP-2010-Kieserman-pdb.prot5427.txt","7","8. After adjustments are made, assemble the modified, individual tiff files into a movie using QuickTime Pro (File Open Image Sequence)."," :AV1"
"CSHP-2010-Kieserman-pdb.prot5427.txt","8","9. (Optional) Resize and/or compress the movies after assembly:"," o"
"CSHP-2010-Kieserman-pdb.prot5427.txt","8","i. To change the size/quality of a movie, use the File Export Options function in QuickTime Pro."," :AV1o"
"CSHP-2010-Kieserman-pdb.prot5427.txt","8","ii. Use the Settings and Size functions in the Options window."," :AV1o"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","1. Prepare poly-L-ornithine-coated plates and solutions."," :AP1"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","2. Euthanize P6 Long-Evans rats using a CO2 chamber."," :AP1a"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","To prevent having more than a few minutes lag time between the euthanasia and removal of the cerebella, con- sider doing this step in two parts."," a"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","3. Decapitate pups. To isolate the cerebella, begin by grasping a pup head at the nose and insert small surgical scissors into the foramen magnum (the aperture at the base of the skull through which the medulla and spinal cord extend). Proceed laterally, then anteriorly through the ear, and curve toward the midline. After reaching the midline from one side, reinsert scissors into the fora- men magnum, and repeat the cutting procedure on the other side."," :AP1ai"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","the aperture at the base of the skull through which the medulla and spinal cord extend"," i"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","The two incisions should meet at the midline."," a"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","4. Using forceps, carefully lift the skull upward, and flip it back until the hindbrain is exposed (Fig. 1A). Make sure that, as the skull is lifted, the brain remains in the base of the skull."," :AP1vx"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","Fig. 1A"," v"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","Make sure that, as the skull is lifted, the brain remains in the base of the skull."," x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","5. If necessary, cut away any bone or other tissues that are obstructing the sides of the cerebellum. Extract the cerebellum with forceps (Fig. 1B), and place it into a dish containing at least 5 mL of HHGN dissection solution."," :AP1r(t)v"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","Fig. 1B"," v"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","See Troubleshooting."," r(t)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","6. After isolation of the cerebella, use the dissection scope and fine forceps to remove the meninges, as well as any large blood vessels or extraneous brain regions that are stuck to the cerebella."," :AP1ax"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","This step should be performed at a steady pace, with a plate of ice underneath the plate of cerebella."," x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","The meninges tend to come off most easily if you start by pulling away residual choroid plexus from the ventral side, where the fourth ventricle was located."," a"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","2","If you are working with two litters, an additional step of trituration is helpful; see Step 11.iv."," ar(11.iv)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","7. Working in the tissue culture hood, decant the dish of cerebella into a 50-mL Falcon tube. Use a pipette to remove most of the HHGN solution, and then add at least 5 mL of fresh HHGN solu- tion. Repeat this for a total of three washes."," :AP1ar(11.iv)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","8. After removing the last HHGN wash, add 5 mL of trypsin-DNase solution, being sure to first mix the solution by pipetting up and down. Place the tube in a 37°C water bath for 10 min. Gently swirl the cerebella every 5 min."," :AP1x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","being sure to first mix the solution by pipetting up and down"," x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","9. Wash three times with HHGN solution as in Step 7."," :AP1r(7)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","as in Step 7"," r(7)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","10. Prepare an aliquot of DNase for trituration by adding 250 μL of DNase to 5 mL of BME in a sepa- rate Falcon tube. Mix by pipetting."," :AP1ar(11.iv)x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","Mix by pipetting"," x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","If you are working with two litters, you will need ~2 mL more of the DNase solution for additional trituration"," a"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","Step 11.iv)"," r(11.iv)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","11. Remove the last HHGN wash, and add the diluted DNase solution from Step 10 to the cerebella. Dissociate by pipetting up and down with a 5-mL pipette as follows:"," :AP1r(10)x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","Avoiding bubbles is critical at this step."," x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","i. For approximately the first 15 rounds of pipetting, do not press the pipette tip against the very bottom of the tube; rather, allow the cerebella to fall apart into smaller chunks."," :AP1r(t)x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","do not press the pipette tip against the very bottom of the tube; rather, allow the cerebella to fall apart into smaller chunks"," x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","See Troubleshooting."," r(t)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","ii. Continue to pipette up and down, but begin gently pressing the pipette tip against the bottom of the tube."," :AP1i"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","After approximately 10 more rounds of pipetting, you will find that the pieces of tissue are not becoming any smaller."," i"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","iii. Press the pipette tip more firmly against the bottom of the tube, and continue the tritura- tion until clumps of tissue are no longer visible."," :AP1"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","iv. To perform an additional trituration, transfer the cell suspension to ice for 5 min, allowing any remaining undetected clumps to sink to the bottom. Transfer the top half of the cell solu- tion to a separate Falcon tube, and triturate the bottom again using fresh DNase solution."," :AP1"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","12. Centrifuge cells at ~200g for 5 min at 4°C."," :AP1r(t)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","See Troubleshooting."," r(t)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","13. Use a pipette to remove the supernatant. Add 5 mL of culture medium for granule neurons, and pipette up and down until a homogeneous mixture is obtained. Add 10 mL more of culture medium incrementally, pipetting up and down after dispensing every 2-3 mL to ensure that the mixture remains homogeneous."," :AP2"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","3","14. Place the cell suspension on ice, and count a small aliquot using a hemacytometer:"," :AP1; AQ1"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","i. Make a 1:10 dilution of cells by adding 100 μL of the cell suspension to 650 μL of culture medium and 250 μL of trypan blue."," :AP1"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","ii. Count healthy, spherical cells with a halo. Do not count cells that take up the trypan blue."," :AQ1er(t)x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","Do not count cells that take up the trypan blue."," x"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","The expected yield is 10-15 million cells per cerebellum."," e"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","See Troubleshooting."," r(t)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","15. Plate cells at the desired density. For morphological assays in 24-well plates with coverslips, use 17- 20 million cells per plate. For biochemical experiments in six-well plates, use 30 million cells per plate. Scale up for larger plates."," :AP1a"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","For morphological assays in 24-well plates with coverslips, use 17- 20 million cells per plate."," a"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","For biochemical experiments in six-well plates, use 30 million cells per plate. Scale up for larger plates."," a"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","16. Maintain cells by adding AraC solution ~24 h after plating cells to a final concentration of 10 μM. On day in vitro 3 (DIV3), add 1 M glucose to a final concentration of 25 mM."," :AP1r(t)"
"CSHP-2008-Bilimoria-pdb.prot5107.txt","4","See Troubleshooting"," r(t)"
"CSHP-2009-Eilers-pdb.prot5201.txt","2","1. Using microloaders, fill the chosen pipettes with 5-10 μL of the appropriate dye-containing solution."," :AP1x"
"CSHP-2009-Eilers-pdb.prot5201.txt","2","Do not use syringe needles to load the pipettes because these may release heavy metals."," x"
"CSHP-2009-Eilers-pdb.prot5201.txt","2","2. Use standard techniques to lower the pipette into the recording chamber containing the chosen slice. Apply positive pressure to prevent dust floating on the surface from entering the pipette."," :AP1x"
"CSHP-2009-Eilers-pdb.prot5201.txt","2","Apply positive pressure to prevent dust floating on the surface from entering the pipette"," x"
"CSHP-2009-Eilers-pdb.prot5201.txt","2","3. Once the pipette tip is in the standard external saline, set the positive pressure to a level that prevents the ejection of too much dye (see Fig. 1A)."," :AP1v"
"CSHP-2009-Eilers-pdb.prot5201.txt","2","see Fig. 1A"," v"
"CSHP-2009-Eilers-pdb.prot5201.txt","2","4. Apply a brief pulse of stronger pressure immediately before seal formation to clean the surface of the cell."," :AP1"
"CSHP-2009-Eilers-pdb.prot5201.txt","3","5. Perform seal formation and establish the whole-cell configuration as per standard electrophysio- logical recordings"," :AP1ir(t)"
"CSHP-2009-Eilers-pdb.prot5201.txt","3","mmediately after the patch is ruptured, the dye will start to diffuse into the cell. The time required to reach a certain cytosolic dye concentration will depend on the mobility of the dye molecule, the dye concentration in the pipette, the series resistance (Rs) of the pipette, and the cell geometry."," i"
"CSHP-2009-Eilers-pdb.prot5201.txt","3","See Troubleshooting."," r(t)"
"CSHP-2010-Allen-pdb.prot5453.txt","2","1. Anesthetize the fly on ice or with CO2."," :AP1aw"
"CSHP-2010-Allen-pdb.prot5453.txt","2","The fly should be left for 20-30 min after mounting if CO2 is used, because occasionally it can affect recordings."," a"
"CSHP-2010-Allen-pdb.prot5453.txt","2","This is not a problem when using ice; however, the fly must be secured in the wax more quickly because recovery from cooling can be quite rapid."," a"
"CSHP-2010-Allen-pdb.prot5453.txt","2","2. Using forceps, transfer the anesthetized fly to the wax by its legs, and mount it into soft wax on a slide or tray with the ventral side down, pushing the legs into the wax to secure the fly."," :AP1"
"CSHP-2010-Allen-pdb.prot5453.txt","2","3. Pull the proboscis outward and push into the wax so that the head lies slightly forward and down on the surface."," :AP1ir(6)"
"CSHP-2010-Allen-pdb.prot5453.txt","2","This step is important because the head needs to be secure and not move when the stimulating electrodes are inserted"," i"
"CSHP-2010-Allen-pdb.prot5453.txt","2","Keeping the head slightly stretched in front of the thorax will also help prevent inadvertent stimulation of the ventral nerve cord."," i"
"CSHP-2010-Allen-pdb.prot5453.txt","2","Step 6)"," r(6)"
"CSHP-2010-Allen-pdb.prot5453.txt","2","4. Pull the wings outward, away from the thorax, and secure. Ensure that the fly cannot move its thorax and that the areas of the DLM and TTM (Fig. 3, dotted areas) are visible and accessible."," :AP1avx"
"CSHP-2010-Allen-pdb.prot5453.txt","2","Ensure that the fly cannot move its thorax and that the areas of the DLM and TTM"," x"
"CSHP-2010-Allen-pdb.prot5453.txt","2","Fig. 3, dotted areas"," v"
"CSHP-2010-Allen-pdb.prot5453.txt","2","If the fly is mounted incorrectly or not securely, it becomes very difficult to obtain recordings, so it is advisable to practice these steps several times before proceeding with the protocol."," a"
"CSHP-2010-Allen-pdb.prot5453.txt","2","Successful recording from the GFS depends on being able to arrange the five micromanipulators so that the electrodes can be placed within several millimeters of each other. It is worth spending some time moving and adjusting these before a preparation is introduced so that minimal adjustment is required when recordings are needed."," :AP1a"
"CSHP-2010-Allen-pdb.prot5453.txt","2","It is worth spending some time moving and adjusting these before a preparation is introduced so that minimal adjustment is required when recordings are needed."," a"
"CSHP-2010-Allen-pdb.prot5453.txt","2","5. Place the ground electrode into the posterior end of the abdomen (Fig. 2C)."," :AP1v"
"CSHP-2010-Allen-pdb.prot5453.txt","2","Fig. 2C"," v"
"CSHP-2010-Allen-pdb.prot5453.txt","2","6. Place the stimulating electrodes through the eyes into the brain (Fig. 2C)."," :AP1iv"
"CSHP-2010-Allen-pdb.prot5453.txt","2","Fig. 2C"," v"
"CSHP-2010-Allen-pdb.prot5453.txt","2","The brain sits at the back of the head capsule, but electrodes pushed in too far may traverse the head capsule and enter the thorax where they may stimulate the ventral nerve cord directly."," i"
"CSHP-2010-Allen-pdb.prot5453.txt","2","7. Give single pulses of 30-60 V for 0.03 msec and check for successful activation of the GFS by looking for movement of the wings and/or TTM muscle on stimulation."," :AP1r(t)"
"CSHP-2010-Allen-pdb.prot5453.txt","2","See Troubleshooting."," r(t)"
"CSHP-2010-Allen-pdb.prot5453.txt","2","8. Fill the glass electrodes for intracellular recordings with GFS saline or 3 M KCl. Place one of the filled glass electrodes into the left (or right) DLM muscle fiber 45a, which is immediately below the cuticle (Fig. 2C; Fig. 3A)."," :AP1r(t)v"
"CSHP-2010-Allen-pdb.prot5453.txt","2","Fig. 2C; Fig. 3A"," v"
"CSHP-2010-Allen-pdb.prot5453.txt","2","See Troubleshooting"," r(t)"
"CSHP-2010-Allen-pdb.prot5453.txt","3","9. Give a single stimulus as in Step 7 and modulate the stimulus strength by varying the voltage to determine the threshold for eliciting a response."," :AP1air(7)r(t)v"
"CSHP-2010-Allen-pdb.prot5453.txt","3","Set the voltage 5-10 V above the determined threshold for the remainder of the experiment."," a"
"CSHP-2010-Allen-pdb.prot5453.txt","3","he response of a good DLM recording is ~50-70 mV and has a latency of ~1.2-1.4 msec"," i"
"CSHP-2010-Allen-pdb.prot5453.txt","3","Fig. 2D"," v"
"CSHP-2010-Allen-pdb.prot5453.txt","3","See Troubleshooting"," r(t)"
"CSHP-2010-Allen-pdb.prot5453.txt","3","10. Place the second intracellular recording electrode in the right (or left) TTM muscle on the contralateral side with respect to the recording electrode for the DLM (Fig. 2C; Fig. 3B)."," :AP1air(t)v"
"CSHP-2010-Allen-pdb.prot5453.txt","3","Fig. 2C; Fig. 3B"," v"
"CSHP-2010-Allen-pdb.prot5453.txt","3","The TTM muscle fibers are much smaller than the DLM muscle fibers and hence it is more difficult to obtain and main- tain a good recording. The response of a good recording from the TTM is ~30-50 mV and has a latency of ~0.8 msec"," i"
"CSHP-2010-Allen-pdb.prot5453.txt","3","Fig. 2D)"," v"
"CSHP-2010-Allen-pdb.prot5453.txt","3","Protocols can be programmed in software such as pCLAMP to capture 10-msec sweeps to collect data."," a"
"CSHP-2010-Allen-pdb.prot5453.txt","3","See Troubleshooting."," r(t)"
"CSHP-2010-Allen-pdb.prot5453.txt","3","11. Once good recordings have been obtained from the TTM and DLM, give 10 single stimuli with an interval of ~5 sec between the stimuli and determine the average response latency for both GF outputs."," :AP1a"
"CSHP-2010-Allen-pdb.prot5453.txt","3","For this step, a separate software protocol that captures 120-msec sweeps can be used to collect the data."," a"
"CSHP-2010-Allen-pdb.prot5453.txt","3","12. Finally, determine the following frequency by giving 10 trains of 10 stimuli at 100 Hz, with an intermittence of ~2 sec between the trains. Calculate the percentage of the total responses. Perform the same assay for trains of stimuli given at 200 Hz and 300 Hz."," :AP1; AQ2"
"CSHP-2010-Allen-pdb.prot5453.txt","3","13. Compare the TTM and DLM response latencies as well as the following frequencies at 100, 200, and 300 Hz between wild-type and mutant flies."," :AI3"
"CSHP-2010-Daul-pdb.prot5460.txt","2","1. Fill the wells of dissection dishes with 200-400 μL of cold Schneider’s insect medium."," :AP1w"
"CSHP-2010-Daul-pdb.prot5460.txt","2","2. Prepare to dissect the larvae by rolling them onto their dorsal side so that the denticle belts are facing upward."," :AP1"
"CSHP-2010-Daul-pdb.prot5460.txt","2","3. Using a pair of forceps, gently grasp the larva just posterior of the midpoint. With a second pair of forceps, grasp the anterior end of the larva at the base of the mouth hooks."," :AP1"
"CSHP-2010-Daul-pdb.prot5460.txt","2","4. Carefully tear the cuticle behind the mouth hooks using a side-to-side motion while slowly drawing the mouthparts out away from the body. The brain will remain attached to the head and be clearly visible among the gut and salivary glands. Remove any excess tissue, but leave the brain attached to the mouth hooks."," :AP1i"
"CSHP-2010-Daul-pdb.prot5460.txt","2","The brain will remain attached to the head and be clearly visible among the gut and salivary glands."," i"
"CSHP-2010-Daul-pdb.prot5460.txt","2","Leaving the brains connected to the mouth hooks will help the brains sink to the bottom of the tube during washing steps below. Moreover, the mouth hooks are dark in color, which makes it easier to see the brains during experimental manipulations."," i"
"CSHP-2010-Daul-pdb.prot5460.txt","2","5. After dissection, place the brains in a 0.5-mL microcentrifuge tube containing cold Schneider’s insect medium."," :AP1x"
"CSHP-2010-Daul-pdb.prot5460.txt","2","Do not let the tissue sit in Schneider’s insect medium for >20 min."," x"
"CSHP-2010-Daul-pdb.prot5460.txt","2","6. Remove the Schneider’s insect medium from the samples."," :AP1"
"CSHP-2010-Daul-pdb.prot5460.txt","2","7. Add 500 μL of fix solution and incubate with rocking for 23 min at room temperature."," :AP1"
"CSHP-2010-Daul-pdb.prot5460.txt","3","8. Quickly wash the brains twice in ~500 μL of PBSTX at room temperature. Wash again in PBSTX twice for 20 min each at room temperature."," :AP1a"
"CSHP-2010-Daul-pdb.prot5460.txt","3","Once fixed, samples can be held in extended washes to synchronize them before proceeding with further processing."," a"
"CSHP-2010-Daul-pdb.prot5460.txt","3","9. Incubate the samples in ~500 μL of block solution (D) for at least 30 min at room temperature."," :AP1"
"CSHP-2010-Daul-pdb.prot5460.txt","3","10. Incubate in primary antibody diluted in PBSBTX for 4 h at room temperature or overnight at 4°C."," :AP1i"
"CSHP-2010-Daul-pdb.prot5460.txt","3","Conditions are dependent on the specific antibody being used. For example, Dpn staining is better when incubated for 3-4 h at room temperature rather than overnight at 4°C."," i"
"CSHP-2010-Daul-pdb.prot5460.txt","3","11. Quickly wash the brains twice in PBSBTX at room temperature. Wash again in PBSBTX twice for 30 min each at room temperature."," :AP1"
"CSHP-2010-Daul-pdb.prot5460.txt","3","12. Incubate the samples in secondary antibody for 1.5 h at room temperature or overnight at 4°C. Protect the samples from light after this point."," :AP1ix"
"CSHP-2010-Daul-pdb.prot5460.txt","3","Protect the samples from light after this point."," x"
"CSHP-2010-Daul-pdb.prot5460.txt","3","Secondary antibodies are typically diluted in PBSBTX."," i"
"CSHP-2010-Daul-pdb.prot5460.txt","3","13. Quickly wash the brains twice in PBSTX at room temperature. Wash again in PBSTX twice for 30 min each at room temperature."," :AP1"
"CSHP-2010-Daul-pdb.prot5460.txt","3","14. Equilibrate the brains in ProLong Gold at room temperature."," :AP1a"
"CSHP-2010-Daul-pdb.prot5460.txt","3","Samples can be stored in the dark at room temperature."," a"
"CSHP-2010-Daul-pdb.prot5460.txt","3","15. Adhere two 22- × 22-mm coverslips to a microscope slide using a small amount of 70% glycerol, leaving an ~5-mm space between them."," :AP1ir(19)"
"CSHP-2010-Daul-pdb.prot5460.txt","3","These coverslips act as spacers to prevent the brains from being deformed by the 24- × 40-mm coverslip"," i"
"CSHP-2010-Daul-pdb.prot5460.txt","3","in Step 19."," r(19)"
"CSHP-2010-Daul-pdb.prot5460.txt","3","16. Transfer the brains to the slide using a pipette with the tip cut off."," :AP1"
"CSHP-2010-Daul-pdb.prot5460.txt","3","17. Using forceps, remove all excess tissue including the optic discs from each brain. Be sure to leave the ventral nerve cord intact, because it will aid in proper orientation of the brain on the slide."," :AP1r(t)x"
"CSHP-2010-Daul-pdb.prot5460.txt","3","Be sure to leave the ventral nerve cord intact, because it will aid in proper orientation of the brain on the slide."," x"
"CSHP-2010-Daul-pdb.prot5460.txt","3","See Troubleshooting."," r(t)"
"CSHP-2010-Daul-pdb.prot5460.txt","4","18. Orient the brains ventral side down."," :AP1i"
"CSHP-2010-Daul-pdb.prot5460.txt","4","If the ventral cord is intact, the brain will sit in the appropriate upright position. Without the ventral cord, it is difficult to keep the brain in the proper position, and it will tend to end up resting on its anterior or posterior surface."," i"
"CSHP-2010-Daul-pdb.prot5460.txt","4","19. Place a 24- × 40-mm coverslip over the samples and backfill the space between the slide and the coverslip by pipetting a small amount of mounting medium along the edge of the coverslip."," :AP1ir(t)"
"CSHP-2010-Daul-pdb.prot5460.txt","4","Backfilling will reduce the formation of air bubbles trapped in the slide."," i"
"CSHP-2010-Daul-pdb.prot5460.txt","4","See Troubleshooting."," r(t)"
"CSHP-2010-Granstedt-pdb.prot5410.txt","2","The procedure must be performed under Biosafety Level 2 conditions."," :X1x"
"CSHP-2010-Granstedt-pdb.prot5410.txt","2","1. Anesthetize the animal by injecting intraperitoneally with 16 μL ketamine/xylazine per gram of body weight."," :AP1a"
"CSHP-2010-Granstedt-pdb.prot5410.txt","2","Wait until the animal is anesthetized by checking paw-pinch withdrawal and corneal reflexes before continuing."," a"
"CSHP-2010-Granstedt-pdb.prot5410.txt","2","2. Lay the animal on its back on a piece of cardboard. Immobilize the limbs with tape. Stretch back the nose by securing a rubber band across the mouth."," :AP1; W3"
"CSHP-2010-Granstedt-pdb.prot5410.txt","2","Monitor the animal during the entire surgical procedure and test the depth of anesthesia regularly (e.g., by controlling respiratory rate and paw-pinch withdrawal reflex)."," W3"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","3. Using an alcohol swab, wet the neck area from the base of the chin to just above the ribcage."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","4. Use a razor blade to shave away the hair, exposing the skin."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","5. Use cotton-tipped applicators to apply disinfectant solution/surgical scrub to the shaved area. Finish by swabbing with 70% ethanol."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","6. Grasp the skin with forceps. Use a sterile scalpel to make a shallow incision ~1.5-cm long from the ribcage to the chin."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","7. Gently detach the glands from the skin using tissue separator scissors."," :AP1i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","The salivary glands are located immediately below the skin, and adhere to it via layers of connective tissue."," i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","8. Rinse the Hamilton syringe with DPBS before drawing up PRV inoculum."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","9. Using the Hamilton syringe, inject 5 μL of the PRV369 into each side of the salivary glands:"," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","i. Inject PRV into one side of the salivary gland by making two to three separate injections in the center."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","ii. Keep the syringe in place for ~10 sec at each injection site."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","iii. Repeat with the other side."," :CL(9.i-ii)1i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","Injecting in both the left and right glands doubles the chance of finding infected cells and reduces the number of animals used."," i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","10. Close the incision with silk sutures. Apply disinfectant solution to the sutured area."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","11. For post-operative analgesia, inject 5 μL of the diluted buprenorphine per gram of body weight intraperitoneally."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","12. Keep the mouse on a heating blanket at 37°C until it recovers. Put the animal in a new cage by itself."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","13. At the desired time post-inoculation, anesthetize the animal for ~10 min in an isoflurane chamber using 2% isoflurane."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","14. Place the animal on its back on the imaging stage. Position the animal’s nose in the isoflurane cone. Tape down all limbs."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","15. Insert the rectal temperature probe. Turn on the heating blanket to keep the animal’s temperature at 37°C."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","16. Under a dissection microscope, use stitch scissors to remove the sutures."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","17. Use tissue-separator scissors to separate the glands from the skin. With the aid of retractors, pull open the skin to better expose the glands."," :AP1i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","Healthy glands have lobules and appear pinkish gray. It is rare to detect any damage in the glands from the first surgery. Even if some inflammation occurs in the glands, it does not affect imaging in the submandibular neurons."," i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","18. Using fine forceps to cut connective tissue, locate the SMG anterior to one of the salivary glands."," :AP1i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","3","The SMGs are located along the salivary duct and surrounded by connective tissue. They appear as small translucent beads. Healthy ganglia should have detectable capillaries with blood flow."," i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","4","19. Lift the ganglion onto a small metal platform that is controlled by a micromanipulator."," :AP1a"
"CSHP-2010-Granstedt-pdb.prot5410.txt","4","Apply some tension so that the ganglion stays in place."," a"
"CSHP-2010-Granstedt-pdb.prot5410.txt","4","20. Move the stage from the dissection microscope to an upright imaging microscope. Perfuse the area with warm mammalian Ringer’s solution."," :AP1"
"CSHP-2010-Granstedt-pdb.prot5410.txt","4","21. Image calcium transients with a one-photon wavelength of 480 nm (or two-photon excitation at 920 nm) and with a 500- to 550-nm bandpass emission filter."," :AV1ir(t)"
"CSHP-2010-Granstedt-pdb.prot5410.txt","4","Labeled cells will have dim background fluorescence that fills the soma compared with nonlabeled cells. Transients are bright and can be seen in the raw movies."," i"
"CSHP-2010-Granstedt-pdb.prot5410.txt","4","See Troubleshooting."," r(t)"