I.
Cell Culture
II.
Crosslinking: Performed by tissue culture
A.
Suspension Cells (or Adherent Cells that have been pelleted)
1.
Cells are ready when they have reached a density of 5-8 x 10 5 cells/ml. Rinse once with PBS and resuspend approx 1 x 10 8 cells in 10ml corresponding plain media (serum-free).
NOTE: Cell viability >90% is desired.
2.
In the hood, add 1ml freshly made 11% Formaldehyde Solution (A1) to a final concentration of 1%. Incubate at RT for 10min on rocker.
3.
Add 1.6ml 1.0M Glycine Solution (A2) to a final concentration of 0.125M to quench the reaction. Incubate at RT for 5min on rocker.
4.
Centrifuge for 5min at 300xg at 4 o C.
5.
Pipet off supernatant and rinse twice with 20ml ice-cold PBS (spin 5min at 300xg at 4 o C).
6.
Can store cell pellet at -80 o C.
B.
Adherent Cells (still on the flask)
1.
Grow cells two days past 90% confluency (ideally want 1 x 10 8 cells). Pour media off flask and rinse once with PBS.
2.
Add 20ml corresponding plain media (serum-free) with 2ml freshly made 11% Formaldehyde
Solution (A1) to a final concentration of 1%. Incubate at RT for 10min on rocker.
NOTE: ChIP works best when cells are healthy but not replicating so the majority of factors are associated with the chromosomes.
3.
Add 3.1ml of 1.0M Glycine Solution (A2) to a final concentration of 0.125M. Incubate at RT for 10min on rocker.
4.
Rinse fixed cells twice with ice-cold PBS.
5.
Add 20ml PBS with 0.84ml 25x Protease Inhibitor Cocktail (final concentration of 1x) and detach cells by scraping.
6.
Centrifuge for 5min at 300xg at 4 o C to pellet cells.
7.
Can store cell pellet at -80 o C.
III.
Sonication via Diagenode sonicator

Ensure Diagenode Chiller is filled with dH20. Turn sonicator on and set chiller to 4 0 C.

Resuspend 1 protease inhibitor cocktail (PIC) tablet in 1ml of ultrapure water to obtain 50X concentrate. PIC is stable for only 1 day but can be re-used up to 1 week if spiked with phenylmethane sulfonyl fluoride (PMSF).
A.
Nuclei preparation
1.
Thaw up to 10 8 cross-linked cells on ice for ~10min.
2.
Determine the amount of nuclei lysis buffer (A3) needed for sample resuspension to a concentration of 20 X 10 6 cells per 0.4 ml of buffer, taking into consideration the initial volume of the cell pellet. Add freshly made 50X PIC and if needed 200X PMSF (if PIC was not freshly made) to a final concentration of 1X.
3.
Resuspend and break up any cell clumps by pipeting up. Incubate for 10min on ice.
4.
Aliquot 0.4ml of sample per 15ml polypropylene tubes.

B.
Chromatin Shearing
1.
Clean Diagenode “tip probes” well with dH20 and 70% ethanol before and after each use.
2.
Screw the probes into the 15ml tube containing your sample to sonicate. Ensure probes are centered within tubes. Allow assembly to incubate on ice for ~3min.
3.
Set metal “O” rings in place on sonicator and place samples in position.
4.
Sonicate samples for 15min twice, using following settings. Check that caps have not come loose after each 15min 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 1.5ml tube. Centrifuge at max speed for 10min at 4C. Transfer supernatant into a labeled 50ml conical tube, taking care not to disturb the pellet.
7.
Add the appropriate buffers to pooled samples per ratio below:

0.1ml sample in Nuclei Lysis Buffer (A3)

0.9ml ChIP Dilution Buffer (A5)

0.5ml RIPA-150 (A7)

28ul 50X PIC

7ul of 200X PMSF (if needed)
8.
Reserve 100ul aliquot for QC purposes and store the rest at -80C. After chromatin shearing efficiency is checked, sample can be split into 100ug aliquots and re-stored at -80C.
9.
If needed reserve a 200ul aliquot for input control library use. Add the following to sample:

275ul of 1XTE and 30ul 5M NaCl

25ul of 20% SDS and 1ul of RNAseA

Incubate for 4hr at 65C

Add 5ul of Proteinse K and continue to incubate O/N at 65C.

Clean via phase-lock tube, EtOH precipitate and resuspend in 100ul of Qiagen EB.

Quantitate sample using Qubit HS kit.
C.
Chromatin Shearing Efficiency Analysis
1.
Add 400ul of PK Digestion Buffer (A6) and 5ul of Fermentase Proteinase K (20mg/ml) to
100ul of sheared chromatin. Heat for at least 2hours to O/N at 55 o C.
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 to bottom of tube.

In the fume hood, aliquot sample into phase lock tube and add an equal volume of phenol/chloroform/isoamyl alcohol to tube. Mix samples well by inversion.

Spin at RT for 5min at 14000g.

Aliquot sample into a new 1.5ml 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 sample for at least 30min at -80C.

Remove sample from freezer and allow thawing for ~5min on benchtop.

Pellet sample by spinning at max speed for 10min at 4C. Remove supernatant carefully.

Wash pellet twice with 1ml of 70% ethanol and spin at 4C for 5 min each.

Carefully remove supernatant and air dry pellet on bench top.

Resuspend pellet in 50ul of elution buffer.

4.
Quantify sample using nanodrop. Calculate concentration for the stock sheared chromatin.
Run 1ug on 1.2% agarose gel at 100V for 45 min to check fragment size (ideally 200-500bp).
IV.
Chromatin Precipitation (ChIP) using 100ug of sheared chromatin equivalent
A.
H3K9me3, H3K27me3, H3K36me3 using protein A/G agarose beads
Day 1 (work on ice and use filtered tips)
Pre-clear chromatin
1.
In low-bind tube, mix 25ul each of protein A and protein G beads (cut off end of tip to aid pipetting; mix bead slurry well before pipetting). Add 1ml cold PBS to mixture. Gently vortex and centrifuge for 3min at 4000rpm at 4 o C.
2.
Pipet off supernatant. Wash 2 more times with cold PBS.
3.
Spin down sheared chromatin for 5min at max speed at 4 o C to pellet any precipitates.
4.
Adjust chromatin volume to 1-1.2ml with ChIP Dilution Buffer and add to rinsed A/G protein bead mix.
5.
Add 10ul normal rabbit serum (rehydrated with 5ml of dH
2
0 and stored in the -20C in 100ul aliquots).
6.
Secure tubes to rocker and pre-clear chromatin O/N at 4 o C.
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 or normal IgG to protein A/G beads
1.
In low-bind tube, mix 15ul each of protein A and protein G beads (cut off end of tip to aid pipetting; mix bead slurry well before pipetting). Add 1ml cold PBS to mixture and centrifuge for 3min at 4000rpm at 4 o C.
2.
Pipet off supernatant. Rinse 2 more times with cold PBS.
3.
Add specific antibody adjusted to 1ml with PBS rinsed A/G protein beads.
Marks
H3K9me3
H3K27me3
Vendor
Millipore
Millipore
Catalog#
07-442
07-449
Amount
4ug/IP
4ug/IP
H3K36me3 Abcam Ab9050 4ug/IP
NOTE: For all 3 H3K… marks above, one IP is sufficient to generate enough ChIP DNA for library construction.
7.
Secure tubes to rocker and pre-bind antibody O/N at 4 o C.
8.
Record catalogue and lot numbers of antibody for future reference.
Day 2
1.
Centrifuge pre-cleared chromatin at 4000rpm for 3min at 4 o C.
2.
Transfer supernatant to new low-bind tube (do not disturb bead pellet). Centrifuge at maximum speed for 5min at 4 o C and save supernatant .
3.
Centrifuge pre-bound antibody-bead complexes at 4000rpm for 3min at 4 o C. Pipet off supernatant without disturbing beads. S ave beads .
4.
Add pre-cleared chromatin (from step 2) to anti-body bead complex and incubate for 2-4 hours at 4 o C on rocker.
5.
Centrifuge at 4000rpm for 3min at 4 o C. Pipet off supernatant without disturbing beads. Save beads.

6.
Perform the following wash steps with 0.8ml COLD buffer. Add buffer to beads and gently vortex to mix. Centrifuge at 4000rpm for 4min at 4 o C. Pipet off supernatant. a.
1 times with RIPA-150 (A7) b.
2 times with RIPA-500 (A8) c.
2 times with RIPA-LiCl (A9) d.
3 times with 1x TE Buffer, pH8.0 (A10)
7.
To elute ChIP DNA from beads, add 100ul freshly made Elution Buffer (A11). Incubate
10min at RT on rocker. Centrifuge for 3 min at 4000rpm at RT. Transfer supernatant to new low-bind tube.
8.
Repeat step 8 with fresh Elution Buffer (final volume is 200ul).
9.
Incubate O/N at 65 o C to reverse crosslinking (mix every ½ hour for the first few hours).
Day 3
1.
Purify the reverse-crosslinked ChIP DNA using a MinElute PCR purification kit. Elute in 2 x
12ul Buffer EB (final volume is 24ul).
B.
CTCF, H3K4me3, Pol II using Dynabeads
Day 1 (work on ice and use filtered tips)
1.
Mix Dynabeads well and aliquot appropriate vol of beads to low-bind tube. See chart below.
Add 1ml cold 1xPBS and flick tube to mix.
2.
Place tube in magnetic stand. Invert several times to mix and allow beads to clump ~1min.
3.
Pipet off PBS. Wash two more times with cold PBS and gently vortex to mix.
4.
Add specific antibody adjusted to 500ul with RIPA-150 (A7) to rinsed Dynabeads.
Note: CTCF and H3K4me3 uses sheep anti-rabbit Dynabeads
Note: Pol II 8WG16 uses sheep anti-mouse Dynabeads
Marks
CTCF
H3K4me3
Dynabeads
100ul/IP Cell Signaling
100ul/IP
Pol II 8WG16 40ul/IP
Vendor
Cell Signaling
Covance
Catalog# Ab Amount
2899 20ul/IP
9751 20ul/IP
MMS-126R 10ul/IP
NOTE: For CTCF, 2-4 IPs are required to generate sufficient ChIP DNA for library construction. For H3K4me3 or Pol II, 1 IP should be enough.
5.
Pre-bind antibody for 6 hours at 4 o C on rocker.
6.
Record catalogue and lot numbers of antibody for future reference.
7.
Place Ab bound Dynabeads in magnetic stand. Invert several times to allow beads to clump and remove supernatant.
8.
If sonicated chromatin is >6mnths old, pellet any precipitate by spinning 100ug of sample at
4 o C for 5min at max speed.
9.
Add chromatin to Ab-bound Dynabeads. Flick tube to mix. Secure tubes on rocker and incubate at 4 o C O/N.
Day 2
1.
Place tube in magnetic stand. Invert several times to allow beads to clump. Remove supernatant without disturbing beads. Save beads.

2.
Perform the following wash steps with 0.8ml COLD buffer. Flick tube to resuspend beads.
Incubate each wash 5min at 4 o C on rocker. Place tube in magnetic stand. Invert several times to mix and allow beads to clump. Pipet off supernatant. a.
1 times with RIPA-150 (A7) b.
2 times with RIPA-500 (A8) c.
2 times with RIPA-LiCl (A9) Aspirate off suds during final wash. d.
2-3 times with 1x TE Buffer, pH8.0 (A10) (Don’t need to incubate on rocker at this step)
Aspirate off suds after final wash.
3.
Resuspend beads in 200ul freshly made Direct Elution Buffer (A12).
4.
Add 1ul RNase A and incubate for a min of 4hrs to O/N at 65 o C to reverse crosslink (vortex every ½ hour for the first few hours).
Day 3
1.
Quick spin sample up to 4000rpm and place in magnetic stand. Transfer supernatant to a new low-bind tube and add Fermentas Proteinase K to a final concentration of 250ug/ml. Incubate for at least one hr to O/N at 65 o C.
2.
Purify the reverse-crosslinked ChIP DNA using phase lock tubes and EtOH precipitation.
Resuspend sample in 25ul of elution buffer.
V.
Library Construction
A.
End-Repair of ChIP DNA fragments
1.
Quantify ChIP DNA using Qubit HS Assay kit. Protocol is optimized for 50ng ChIP input
DNA, but amount may very likely be less.
2.
Prepare the following reaction mix using Epicentre End-It DNA End Repair kit (ER0720):
ChIP DNA (5-50ng) 34ul
End It 10X buffer
End It dNTP mix
End It ATP
End-Repair Enzyme Mix
5ul
5ul
5ul
1ul
Total Volume 50ul
3.
Incubate at RT for 45min.
4.
Purify with Qiagen MinElute column using Buffer PB. Elute in 16ul of Buffer EB twice.
B.
Addition of Adenine to 3’ ends of purified DNA fragments
1.
Prepare the following reaction mix:
Blunt-ended DNA 32ul
10X NEB Buffer 2 5ul
1mM dATP(Fermentas #R0141)
Klenow (3’-5’ exo-)(5U/ul)
1.
Prepare the following reaction mix:
A-tailed DNA
10ul
3ul (NEB #M0212L)
Total Volume 50ul
2.
Incubate for 30min at 37 o C water bath.
3.
Purify with Qiagen MinElute column using Buffer PB. Elute in 10ul of Buffer EB.
C.
Ligation of Adapters (Overnight reaction)
10ul ddH
2
O 8ul

Adapter oligo mix (1:20 dil) 1ul
T
4
DNA Ligase Buffer
400U/ul T
4
DNA Ligase
3ul
3ul
Total Volume 25ul
2.
Incubate for 30min at 20 o C in cooling block.
3.
Transfer to 16 o C O/N.
4.
Purify on one MinElute column using Buffer PB. Elute in 20ul of Buffer EB.
Ligation of Adapters (NEB Quick Ligation Kit M2200L)
1.
Prepare the following reaction mix:
A-tailed DNA 10.0ul
2X Ligase Buffer
Adapter oligo mix (1:10 in H20)
15.0ul
0.5ul
Quick Ligase Enzyme dH20
Total Volume
1.5ul
3.0ul
30.0ul
2.
Incubate for 15min at RT.
3.
Purify on one MinElute column using Buffer PB. Elute in 10ul of Buffer EB twice.
4.
Store excess post-ligation product at -20 o C.
D.
Enrichment PCR
1.
Prepare the following PCR reaction mix:
Ligated DNA 5ul
Phusion HF DNA polymerase
PCR primer PE 1.0 (1:2 dil)
25ul (NEB #F531L)
1ul (PE-102-1004 see PO#687438)
PCR primer PE 2.0 (1:2 dil) dH20
Total Volume
1ul
18ul
50ul
2.
Amplify using the following PCR protocol: a.
30” at 98 o C b.
[10”at 98 o C, 30” at 65 o C, 30” at 72 o C] for 16 cycles c.
5min at 72 o C d.
Hold at 4 o C
3.
Store at -20 o C.
E.
Final Fractionation and Clean-up
1.
Load entire enrichment product on a 2% agarose gel. Split sample into two lanes.
2.
Purify by cutting out darkest band from gel. Pool gel slices.
3.
Use the MinElute gel purification kit protocol with the following modifications to purify library.
4.
Add 6x volume of Buffer QG and allow gel slice to melt at RT for 30min with occasional vortexing.
5.
Add 2X volume of Isopropanol and mix.
6.
Load sample on a QIAGEN MinElute column with syringe adaptor and let sit for 15min to bind sample to column.
7.
Wash with 500ul Buffer QG.
8.
Add 750ul Buffer PE and let sit for five minutes.
9.
Do two more washes with Buffer PE. Spin to dry column.
10.
Add 20ul Buffer EB to column and let sit five minutes. Elute in low-bind tube.
11.
Repeat previous step with same eluate (final volume is 20ul).
12.
Run 8ng of sample on a 3% agarose gel to verify size fragments.
13.
Quantify on Qubit using HS kit.

Buffers and Solutions : Filter solutions
A1. Formaldehyde (8.8%HCHO, 50mM Tris-HCl pH8, 0.1M NaCl, 1mM EDTA pH8)
1.
37% HCHO 1200 ul
2.
1.0M Tris-HCl pH8
3.
5M NaCl pH8
250 ul
100 ul
4.
0.5M EDTA pH8
5.
ddH
2
0
6.
Total Vol
10 ul
3.44 ml
5 ml
Ambion #9262
A2. 1.0M Glycine Solution
1.
Glysine
2.
ddH
2
0
3.
Total Vol
3.76 g
50 ml
50 ml
A3. Nuclei Lysis Buffer (50mM Tris-HCl pH8, 10mM EDTA pH8, 1% SDS) : Store at RT
1.
1.0M Tris-HCl pH8 2.5 ml
2.
0.5M EDTA pH8
3.
20% SDS
1 ml
2.5 ml
4.
ddH
2
0
5.
Total Vol
44 ml
50 ml
A5. ChIP Dilution Buffer (50mM Tris-HCl pH8, 0.167M NaCl, 1.1% Triton X-100, 0.11% sodium deoxycholate) :
Store at 4C
1.
1.0M Tris-HCl pH8
2.
5M NaCl pH8
2.5 ml
1.67 ml
3.
10% Triton X-100 5.5 ml
4.
10% sodium deoxycholate 0.55 ml
5.
ddH
2
0
6.
Total Vol
39.78 ml
50 ml
Sigma # T9284
Sigma # D6750
A6. PK digestion Buffer (10mM Tris-HCl pH8, 1mM EDTA pH8, 0.5% SDS) : Store at RT
1.
1.0M Tris-HCl pH8 0.5 ml
2.
0.5M EDTA pH8
3.
20% SDS
4.
ddH
2
0
5.
Total Vol
0.1 ml
1.25 ml
48.15 ml
50 ml
A7. 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.
1.0M Tris-HCl pH8
2.
5M NaCl pH8
3.
0.5M EDTA pH8
2.5 ml
1.5 ml
0.1 ml
4.
20% SDS 0.25 ml
5.
10% Triton X-100 5 ml
6.
10% sodium deoxycholate 0.5 ml
7.
ddH
2
0
8.
Total Vol
40.15 ml
50 ml

A8. 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.
1.0M Tris-HCl pH8
2.
5M NaCl pH8
3.
0.5M EDTA pH8
4.
20% SDS
2.5 ml
5 ml
0.1 ml
0.25 ml
5.
10% Triton X-100 5 ml
6.
10% sodium deoxycholate 0.5 ml
7.
ddH
2
0
8.
Total Vol
36.65 ml
50 ml
A9. RIPA-LiCL (50mM Tris-HCl pH8, 1mM EDTA pH8, 1% Nonidet P-40, 0.7% sodium deoxycholate, 0.5M
LiCl2) : Store at 4C
1.
1.0M Tris-HCl pH8 2.5 ml
2.
0.5M EDTA pH8
3.
10% Nonidet P-40
0.1 ml
5 ml
4.
10% sodium deoxycholate 3.5 ml
5.
7.5M LiCl2
6.
ddH
2
0
7.
Total Vol
3.3ml
35.6 ml
50 ml
Roche # 11332473001
VWR # 101175-850
A10. 1 X TE Buffer pH8.0 (10mM Tris-HCl pH8, 1mM EDTA pH8) : Store at 4C
1.
1.0M Tris-HCl pH8 0.5 ml
2.
0.5M EDTA pH8
3.
ddH
2
0
0.1 ml
49.4 ml
50 ml 4.
Total Vol
A11. Elution Buffer (1% SDS, 0.1M NaHCO3, 10% proteinase K) : Make fresh each use
1.
20% SDS 5 ul
2.
1M sodium bicarbonate
3.
Proteinase K
4.
ddH
2
0
5.
Total Vol
10ul
10 ul
75 ul
100 ul
Fermentas #EO-0491
A12. Direct Elution Buffer (10mM Tris-HCl pH8, 0.3M NaCl, 5mM EDTA pH8, 0.5%SDS) : Make fresh each use
1.
1.0M Tris-HCl pH8 0.1 ml
2.
5M NaCl
3.
0.5M EDTA pH8
0.6 ml
0.1 ml
4.
20% SDS
5.
ddH
2
0
6.
Total Vol
0.25 ml
8.95 ml
10 ml