ChIP-Seq Protocol: Cells
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. Use within 1 to 2 days.
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.
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 any cell clumps by gently pipetting up and down. Incubate for 10min on ice.
5. Aliquot 0.4ml of sample into one 15ml falcon polystyrene tube and set on ice.
B. Chromatin Shearing
1. Clean Diagenode “tip probes” 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 samples for 10min twice, using following settings. Check that caps have not come loose after each 10min 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 5-10 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
· 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 to O/N at 65C : 275ul of 1xTE buffer, 30ul 5M NaCl, 25ul of 20% SDS and 1ul of RNAseA
· Allow samples to cool slightly and add 10ul of Proteinse K. Incubate 1hr to 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 1hr to O/N at 55oC.
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 Precipitation (ChIP) using ~60ug of sheared chromatin equivalent
A. CTCF/Histone mods: 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 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 AmountCTCF / 60ul/IP / Cell Signaling / 2899B / 12ul/IP
H3K4me3 / 60ul/IP / Cell Signaling / 9751B / 12ul/IP
H3K27ac / 48ul/IP / Active Motif / 39133 / 3ug/IP
NOTE: For CTCF, more than 1 IP may be required to generate enought 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 60ug 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 and 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 or O/N 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 for 1-2hrs at 55C.
Day 3
1. Purify the reverse-crosslinked ChIP DNA 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.
B. Histone mods: protein A/G agarose beads (Millipore #16-156 & #16-266)
Day 1 (work on ice and use filtered tips)
Pre-clear chromatin
1. Mix protein bead slurry well. Use a wide bore tip to aid in pipetting.
2. Per IP, mix 25ul each of protein A and protein G beads in a low bind tube. Add 1ml cold 1X PBS and gently mix. Centrifuge at 3000rpm for 3min at 4C.
3. Carefully discard supernatant. Wash 2 more times with cold 1X PBS.
4. If sonicated chromatin is >3 months old, pellet any precipitate by spinning sample at max speed for 5min at 4C.
5. Add 60ug of chromatin to A/G protein bead mix. Adjust vol to 1ml with RIPA-150 if needed.
6. Add 6ul normal rabbit serum (rehydrated with 5ml of dH20 and stored at -20C in 100ul aliquots).
7. Secure tubes to rocker and pre-clear chromatin O/N at 4C.
NOTE: Binding capacity for protein A agarose beads is 40mg human IgG/ml agarose. Binding capacity of protein G agarose beads is 20mg human IgG/ml agarose.
Pre-bind Ab to protein A/G beads
1. Mix protein bead slurry well. Use a wide bore tip to aid in pipetting.
2. Per IP, mix 15ul each of protein A and protein G beads in a low bind tube. Add 1ml cold 1X PBS and gently mix. Centrifuge at 3000rpm for 3min at 4C.
3. Carefully discard supernatant. Wash 2 more times with cold 1X PBS.
4. Add specific antibody adusted to 1 ml with RIPA-150 to A/G protein beads.
Marks / Vendor / Catalog# / AmountH3K4me1 / Active Motif / AM39297 / 3ul/IP
H3K9me3 / Active Motif / AM39161 / 3ug/IP
H3K27me3 / Millipore / 07-449 / 2.4ug/IP
H3K36me3 / Active Motif / AM61101 / 6ul/IP
5. Secure tubes to rocker and pre-bind Ab O/N at 4C.
6. Record catalogue and lot numbers of Ab for future reference.
Day 2
1. Centrifuge pre-cleared chromatin at 3000rpm for 3min at 4C.
2. Transfer supernatant to new low-bind tube (do not disturb bead pellet). Centrifuge at max speed for 5min at 4C and save supernatant.
3. Centrifuge pre-bound Ab-bead complex at 3000rpm for 3min at 4C. Discard supernatant without disturbing beads. Save beads.
4. Add pre-cleared chromatin (from step 2) to Ab-bead complex. Incubate for 4 hrs at 4C on rocker.
5. Centrifuge at 3000rpm for 3min at 4oC. Discard supernatant. Save beads.
6. Perform the following wash steps with 0.8ml of COLD buffer. Add buffer to beads and mix for 10min at 4C. Centrifuge at 3000rpm for 3min at 4C. Pipet off supernatant.
· 1 times with RIPA-150
· 2 times with RIPA-500
· 2 times with RIPA-LiCl
· 2 times with 1x TE Buffer, pH8.0
7. Add 160ul of freshly made Elution Buffer to elute DNA. Place on rocker and incubate for 10min at RT. Centrifuge at RT for 3 min at 3000rpm and transfer 150ul supernatant to a new low-bind tube.
8. Repeat step 7 with 150ul of fresh Elution Buffer (final volume is 300ul).
9. Add 18ul of 5M NaCl and 1ul of RNase A.
10. Incubate O/N at 65C to reverse crosslink (briefly mixing every ½ hour for the first few hours).
11. Add 3ul of Proteinase K and incubate for 1-2hrs at 55C.
Day 3
1. Purify the reverse-crosslinked ChIP DNA using phase-lock tubes and EtOH precipitation.
2. Resuspend sample in 25ul of 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. Phenol/Chloroform/Isoamyl Alcohol (25:24:1 Mixture, pH 6.7/8.0, Liq.): Fisher #BP1752I100
4. Diagenode Bioruptor Sonicator
5. Glycogen: Roche #10901393001
6. Magnetic separation stand: Invitrogen # 123-21D
7. Nanodrop
8. Phenylmethane sulfonyl fluoride (PMSF): Sigma #93482
9. Protease inhibitor cocktail-EDTA-Free (PIC): Roche #11873580001
10. Proteinse K: Fisher #EO0491
11. Rabbit Serum: Jackson ImmunoResearch #011-000-120
12. RNase A: Fisher #NC9499959
13. 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. 1M Sodium Bicarbonate (NaHCO3): Filter using 0.2-0.45 micron filter : Store at RT
· Sodium bicarbonate 2.10g
· ddH20 25ml
Caution: Sodium Bicarbonate is irritating to the lungs
4. 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
5. 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
6. 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
7. 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
8. 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
9. 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
10. 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
11. Elution Buffer (1% SDS, 0.1M NaHCO3): For Agarose beads: Make fresh each use
· 20% SDS 250 ul
· 1M sodium bicarbonate 500 ul
· ddH20 4.25 ml
· Total Vol 5ml
12. Direct Elution Buffer (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA pH8, 0.5%SDS): Dynal beads: 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