ChIP-Seq Protocol
I. Sonication via Diagenode sonicator Ensure Diagenode Chiller is filled with dH20. Turn sonicator on and set chiller to 4C. Resuspend 1 protease inhibitor cocktail (PIC) tablet in 1ml of ultrapure water to obtain a 50X concentrate. PIC is stable for 1 day but can be used up to 1 week if sample is spiked with phenylmethane sulfonyl fluoride (PMSF).
A. Nuclei preparation 1. Thaw cross-linked cells on ice for ~10min. 2. Add nuclei lysis buffer (NLB) to cells to achieve a concentration of ~20 X 106 cells per 0.4 ml of buffer, taking into consideration the initial volume of the cell pellet. 3. Add 50X PIC to a final concentration of 1X (and if needed 200X PMSF). Add 1M sodium butyrate to a final concentration of 10mM. 4. Resuspend and break up any cell clumps by gently pipeting. Incubate for 10min on ice. 5. Aliquot 0.4ml of sample into one 15ml Falcon polypropylene tubes.
B. Chromatin Shearing 1. Clean Diagenode “tip probes” well with 70% ethanol and dH20 before and after each use. 2. Screw the probes into the 15ml tube containing the sample to sonicate. Ensure probes are centered within tubes and incubate on ice for ~3min. 3. Set metal “O” rings in place on sonicator machine and set samples in position. 4. Sonicate twice for 15min, using settings below. Ensure caps have not come loose after each shearing interval. On = 0.5 min Off = 0.5 min Setting = “H”
5. Sonicated sample should appear transparent in color. Samples can be further sonicated an additional 5-15 min if needed. 6. Aliquot samples into a 1.7ml tube. Centrifuge at max speed for 10min at 4C. Transfer supernatant to a labeled conical tube, taking care not to disturb the pellet. 7. Per 0.1ml of sonicated sample in NLB, add the following buffers per ratio below: 0.9ml ChIP Dilution Buffer (CDB) 0.5ml RIPA-150 28ul 50X PIC 7ul of 200X PMSF (if needed) 14ul of 1M sodium butyrate
8. Reserve 100ul of sample for QC purposes and store the rest at -80C. After chromatin shearing efficiency is checked, samples can be stored as 1.4ml aliquots at -80C. 9. If needed, reserve a 200ul aliquot for input control library use. Add the following to input control and incubate for 4hrs at 65C: 275ul of 1xTE buffer, 30ul 5M NaCl, 25ul of 20% SDS and 1ul of RNAseA. Allow samples to cool slightly and add 5ul of Proteinse K. Incubate O/N at 55C. Clean using phenol/chloroform extraction and phase-lock tubes (see below for procedure). EtOH precipitate and resuspend in 100ul of Qiagen EB. Quantify using Qubit HS kit. C. Chromatin Shearing Efficiency Analysis (QC) 1. Add 400ul of PK Digestion Buffer and 5ul of Proteinase K to 100ul of sheared chromatin. Incubate for at least 2hours to O/N at 55C. 2. Clean samples via phenol/chloroform extraction using phase lock tubes. Spin a 2ml phase lock tube at RT for 30sec at 14000g to pellet gel. In the fume hood, aliquot sample into phase lock tube and add an equal volume of phenol/chloroform/isoamyl alcohol. Mix well. Spin at RT for 5min at 14000g. Aliquot sample into a new 1.7ml tube and prepare for ethanol precipitation.
3. Ethanol precipitation. Add 2X volume of 100% ethanol, 0.1X volume of 3M sodium acetate, and 1ul of glycogen to sample. Incubate at -80C for at least 30min. Remove sample from freezer and briefly thaw. Pellet sample by spinning at max speed for 10min at 4C. Remove supernatant carefully. Wash pellet with 1ml of cold 70% ethanol. Spin at max speed for 5 min at 4C. Carefully remove supernatant and repeat wash step. Carefully remove supernatant and allow pellet to dry. Resuspend pellet in 50ul of elution buffer.
4. Quantify sample using nanodrop. Calculate stock sheared chromatin concentration. 5. Check fragment size by running 1ug of sample on a 1.2% agarose gel at 100V for 45min (ideally between 100-500bp).
II. Chromatin Immunoprecipitation using 100ug of sheared chromatin equivalent
CTCF & Histone mods using Invitrogen M-280 sheep anti-rabbit Dynabeads (Invitrogen #112-04D) Day 1 (work on ice and use filtered tips) 1. Mix Dynabeads well. Per chart below, aliquot appropriate vol of beads to PGC low-bind tubes. 2. Add 1ml of cold 1X PBS to beads and gently vortex to mix. 3. Place tube in magnetic stand. Invert several times to mix. Allow beads to clump for ~1min. 4. Pipet off 1X PBS. Repeat wash step two more times. 5. Add specific Ab adjusted to 500ul with RIPA-150 to rinsed Dynabeads.
Marks Dynabeads Ab Vendor Ab Catalog# Ab Amount CTCF 100ul/IP Cell Signaling 2899B 20ul/IP H3K4me 60ul/IP Abcam ab8895 1ug/IP H3K4me3 100ul/IP Cell Signaling 9751B 20ul/IP H3K9me3 Abcam ab8898 H3K27ac 80ul/IP Active Motif 39134 5ug/IP H3K27me3 80ul/IP Millipore 07-449 4ug/IP H3K36me3 80ul/IP Abcam ab9050 4ug/IP NOTE: For CTCF, more than 1 IP may be required to generate ChIP DNA for lib construction.
6. Pre-bind Ab for 6 hours at 4C on rocker. 7. Record catalogue and lot numbers of Ab for future reference. 8. Place Ab-bound Dynabeads in magnetic stand. Invert several times. Allow beads to clump and discard supernatant. Keep beads on ice. 9. If sonicated chromatin is >3 months old, pellet any precipitate by spinning sample at max speed for 5min at 4C. 10. Add 100ug of chromatin to Ab-bound Dynabeads. Gently mix and place on rocker O/N at 4C.
Day 2 1. Place tube in magnetic stand. Invert several times. Allow beads to clump. Discard supernatant. 2. Perform the following wash steps with 0.8ml of COLD buffer. Flick tubes to resuspend beads. Incubate each wash for 5min on rocker at 4C. Place tube in magnetic stand. Invert several times. Allow beads to clump and discard supernatant. 1 times with RIPA-150 2 times with RIPA-500 2 times with RIPA-LiCl. Aspirate suds after second wash. 2 times with 1xTE Buffer, pH8.0. Don’t need to incubate on rocker at this step. Aspirate suds after final wash.
3. Resuspend beads in 200ul freshly made Direct Elution Buffer. 4. Add 1ul of RNase A and incubate for 4hrs at 65C to reverse crosslink (briefly vortex sample every ½ hour for the first few hours). 5. Quick spin sample. Place in magnetic stand. Allow beads to clump and transfer supernatant to a new low-bind tube. 6. Add 3ul of Proteinase K and incubate O/N at 55C.
Day 3 1. Purify the reverse-crosslinked sample using phase lock tubes and EtOH precipitation. 2. Resuspend sample in 25ul of Qiagen elution buffer. 3. Quantify using Qubit HS kit and proceed to library construction.
Material 1. 1.7ml siliconized PP tubes: PGC Scientific #16-8110-09 2. 2ml Heavy Phase Lock Tubes: Fisher #FP2302830 3. Diagenode Sonicator 4. Glycogen: Roche #10901393001 5. Magnetic separation stand: Invitrogen # 123-21D 6. Nanodrop 7. Phenylmethane sulfonyl fluoride (PMSF): Sigma #93482 8. Protease inhibitor cocktail-EDTA-Free (PIC): Roche #11873580001 9. Proteinse K: Fisher #EO0491 10. RNase A: Fisher #NC9499959 11. Qubit HS kit: Invitrogen #Q32854
Buffers and Solutions : Filter solutions
1. 1M Sodium Butyrate (100X): Filter using 0.2-0.45 micron filter : Store at 4C Sodium butyrate 5.5g Sigma # B5887
ddH20 50ml Caution: Sodium butyrate is irritating to the lungs
2. 10% Sodium Deoxycholate: Filter using 0.2-0.45 micron filter : Store at RT Sodium deoxycholate 2g
ddH20 20ml Caution: Sodium deoxycholate is irritating to the lungs
3. Nuclei Lysis Buffer (NLB): (50mM Tris-HCl pH8, 10mM EDTA pH8, 1% SDS) : Store at RT 1.0M Tris-HCl pH8 2.5 ml 0.5M EDTA pH8 1 ml 20% SDS 2.5 ml
ddH20 44 ml Total Vol 50 ml
4. ChIP Dilution Buffer (CDB): (50mM Tris-HCl pH8, 0.167M NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate) : Store at 4C 1.0M Tris-HCl pH8 2.5 ml 5M NaCl pH8 1.67 ml 10% Triton X-100 5.5 ml Sigma # T9284 10% sodium deoxycholate 0.55 ml Sigma # D6750
ddH20 39.78 ml Total Vol 50 ml
5. PK digestion Buffer (10mM Tris-HCl pH8, 1mM EDTA pH8, 0.5% SDS) : Store at RT 1.0M Tris-HCl pH8 0.5 ml 0.5M EDTA pH8 0.1 ml 20% SDS 1.25 ml
ddH20 48.15 ml Total Vol 50 ml
6. RIPA-150 (50mM Tris-HCl pH8, 0.15M NaCl, 1mM EDTA pH8, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate) : Store at 4C 1.0M Tris-HCl pH8 2.5 ml 5M NaCl pH8 1.5 ml 0.5M EDTA pH8 0.1 ml 20% SDS 0.25 ml 10% Triton X-100 5 ml 10% sodium deoxycholate 0.5 ml
ddH20 40.15 ml Total Vol 50 ml
7. RIPA-500 (50mM Tris-HCl pH8, 0.5M NaCl, 1mM EDTA pH8, 0.1% SDS, 1% Triton X-100, 0.1% sodium deoxycholate) : Store at 4C 1.0M Tris-HCl pH8 2.5 ml 5M NaCl pH8 5 ml 0.5M EDTA pH8 0.1 ml 20% SDS 0.25 ml 10% Triton X-100 5 ml 10% sodium deoxycholate 0.5 ml
ddH20 36.65 ml Total Vol 50 ml
8. RIPA-LiCL (50mM Tris-HCl pH8, 1mM EDTA pH8, 1% Nonidet P-40, 0.7% sodium deoxycholate, 0.5M LiCl2) : Store at 4C 1.0M Tris-HCl pH8 2.5 ml 0.5M EDTA pH8 0.1 ml 10% Nonidet P-40 5 ml Roche # 11332473001 10% sodium deoxycholate 3.5 ml 7.5M LiCl2 3.3ml VWR # 101175-850
ddH20 35.6 ml Total Vol 50 ml
9. 1 X TE Buffer pH8.0 (10mM Tris-HCl pH8, 1mM EDTA pH8) : Store at 4C 1.0M Tris-HCl pH8 0.5 ml 0.5M EDTA pH8 0.1 ml
ddH20 49.4 ml Total Vol 50 ml
10. Direct Elution Buffer (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA pH8, 0.5%SDS) : Make fresh each use 1.0M Tris-HCl pH8 0.1 ml 5M NaCl 0.6 ml 0.5M EDTA pH8 0.1 ml 20% SDS 0.25 ml
ddH20 8.95 ml Total Vol 10 ml