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.
6.
7.
8.
9.
Sonicated sample should appear transparent in color. Samples can be further sonicated an
additional 5-10 min if needed.
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.
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
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.
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.
5.
Quantify sample using nanodrop. Calculate stock sheared chromatin concentration.
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 Amount
CTCF
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.
7.
8.
9.
Pre-bind Ab for 6 hours at 4C on rocker.
Record catalogue and lot numbers of Ab for future reference.
Place Ab-bound Dynabeads in magnetic stand. Invert several times. Allow beads to clump and
discard supernatant. Keep beads on ice.
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.
4.
5.
6.
Resuspend beads in 200ul freshly made Direct Elution Buffer.
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).
Quick spin sample. Place in magnetic stand. Allow beads to clump and transfer supernatant to a
new low-bind tube.
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.
5.
6.
Marks
Vendor
Catalog#
Amount
H3K4me1
Active Motif
AM39297
3ul/IP
H3K9me3
Active Motif
AM39161
3ug/IP
H3K27me3
Millipore
07-449
2.4ug/IP
H3K36me3
Active Motif
AM61101
6ul/IP
Secure tubes to rocker and pre-bind Ab O/N at 4C.
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.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
1.7ml siliconized PP tubes: PGC Scientific #16-8110-09
2ml Heavy Phase Lock Tubes: Fisher #FP2302830
Phenol/Chloroform/Isoamyl Alcohol (25:24:1 Mixture, pH 6.7/8.0, Liq.): Fisher #BP1752I100
Diagenode Bioruptor Sonicator
Glycogen: Roche #10901393001
Magnetic separation stand: Invitrogen # 123-21D
Nanodrop
Phenylmethane sulfonyl fluoride (PMSF): Sigma #93482
Protease inhibitor cocktail-EDTA-Free (PIC): Roche #11873580001
Proteinse K: Fisher #EO0491
Rabbit Serum: Jackson ImmunoResearch #011-000-120
RNase A: Fisher #NC9499959
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