Cell Growth and harvest:
- Grow mouse ES cells to two 70% confluent 15 cm culture dishes (a total of 50 · 10e6 cells).
- Crosslink cells by adding 1/37 volume of Formaldehyde 37% (Merck) to each plate.
- Incubate on shaking table for 20 min at RT.
- Quench Formaldehyde by adding 1/7 volume of freshly prepared Glycine 1M.
- Wash cells 2x with 10 ml cold PBS (Lonza).
- Add 10 ml cold PBS and scrape off the cells.
- Spin down cells at 2000g for 10 min at 4°C.
- Discard supernatant, flash freeze pellet in liquid nitrogen, store at -80°C.
Chromatin Extraction:
- Wash pellet with 15 ml buffer A for 10 min at 4°C.
- Spin down at 1300 rpm for 10 min at 4°C.
- Wash pellet with 15 ml buffer B for 10 min at 4°C.
- Spin down at 1300 rpm for 10 min at 4°C.
- Resuspend nuclei in 1 ml buffer C in a 15 ml tube.
Sonication:
- Sonicate samples (Biorupter, Diagenode) 20 times on high power, 30 sec on/ 30 sec off. Keep samples at 4°C.
- Run 10 µl sample (de-crosslink by adding 1µl ProtK (5 mg/ml) and 1µl RNAse (10 mg/ml), 1hr at 37°C) on a 1% agarose gel to check sonication-efficiency (product sizes must be mostly between 400 and 1000 bp).
- Spin down samples at max speed for 10 min. at 4°C.
- Flash-freeze supernatant in dry ice.
Preparation of Beads (Prot A/G PLUS-Agarose beads, Santa Cruz, sc-2003):
- Take 40 µl beads per IP (25% solution).
- Spin down for 1 min at 1000g in 4°C (check for presence of pellet).
- Wash in 750 µl 1X incubation buffer (1X PIC, 0,1% Bovine Serum Albumin (Merck)) at 4°C.
- Repeat previous step 2 times.
- Resuspend beads in 20 µl 1X incubation buffer (1X PIC, 0,1% BSA) at 4°C to get a 50% suspension.
- Incubate and rotate at 4°C overnight.
ChIP Incubation:
- Prepare the following mix (per ChIP) at 4°C:
o 280 µl total volume:
o 107 µl H2O
o 60 µl 5X incubation buffer
o 3 µl 10% BSA
o 10 µl 50X PIC
o 100 µl chromatin suspension
- Add 4 µg antibody, fill to 300 µl with H2O .
- Incubate with rotation at 4°C overnight.
We have used this protocol with the following antibodies:
TAF1: ab51540 (Abcam)
TBP: ab818 (Abcam)
Nanog: A300-397A (Bethyl)
Oct4: Sc-8628 (Santa Cruz)
H3: ab1791 (Abcam)
H3K4Me3: ab8580 (Abcam)
RNA polII: 8WG16
HA: 12CA5
ChIP Wash and pull-down:
- Add 20 µl beads suspension (50%) to each sample.
- Pellet beads at 1000g for 1 min at 4°C.
- Wash beads 2x with buffer 1, each time with a 5 min incubation at 4°C whilst rotating.
- Wash beads 1x with buffer 2, with a 5 min incubation at 4°C whilst rotating.
- Wash beads 1x with buffer 3, with a 5 min incubation at 4°C whilst rotating.
- Wash beads 2x with buffer 4, each time with a 5 min incubation at 4°C whilst rotating.
- After last wash, take out as much supernatant as possible, and elute by adding 400 µl elution buffer. Rotate for 30 min at RT.
- Spin down sample at maximum speed for 1 min.
- Sample is now in supernatant. Take off supernatant without taking any of the beads! Transfer to new tube.
- Get 50 µl input chromatin sample and add elution buffer to same level as other samples.
- Add 16 µl of a 5M NaCl (Sigma) solution to the ChIP eluates and the input chromatin and incubate overnight at 65°C for decrosslinking.
DNA purification:
- Add 1µl RNAse (10 mg/ml) and incubate for 2 hrs at 37°C.
- Add 150 µl Glycogen solution (20 mg/ml) and 50 µl ProtK 5 mg/ml (Roche) and incubate for 2 hrs at 37°C.
- Extract DNA with 600 µl Phenol/Chloroform (Sigma Aldrich) by vortexing for 15 sec.
- Incubate whilst shaking at RT for 5 min.
- Spin down for 5 min at max speed.
- Take off waterphase and transfer to new tube.
- Repeat previous step once.
- Add 600 µl Chloroform/Isoamylalcohol (Sigma Aldrich) to the waterphase.
- Incubate whilst shaking at RT for 5 min.
- Spin down for 5 min at max speed.
- Take off waterphase and transfer to a new 2 ml tube.
- Add 1/10 volume of 3M NaAc pH 5.2, 2.5 vol. 96% EtOH and mix well.
- Precipitate overnight at - 80°C.
- Spin down for 30 min at 13,000 rpm at 4°C
- Discard supernatant carefully, leaving the pellet untouched.
- Wash pellet with 80% EtOH, spin down for 5 min at 13,000 rpm at 4°C.
- Take off supernatant even more carefully, leaving the pellet untouched.
- Dry pellet for a few min in the heat block at 37°C.
- Resuspend in 100-200 µl 10 mM Tris, pH 8.0.
Subsequently, samples can be measured by qPCR.
Buffer compositions:
Buffer A (50 ml):
- 1 ml 1M HEPES-KOH (Sigma) buffer, pH 7.5 (20 mM final)
- 1 ml 0.5M EDTA pH 8.0 (Merck) (10 mM final)
- 50 µl 0.5M EGTA pH 8.0 (Sigma) (0.5 mM final)
- 625 µl Triton X-100 20% (Merck) (0.25% final)
Buffer B:
- 2.5 ml 1M HEPES-KOH buffer, pH 7.5 (50 mM final)
- 1.5 ml 5M NaCl (150 mM final)
- 100 µl 0.5M EDTA pH 8.0 (1 mM final)
- 50 µl 0,5M EGTA pH 8.0 (0.5 mM final)
Buffer C: Note: make this buffer just before use
- 1 ml 1M HEPES-KOH buffer, pH 7.5 (20 mM final)
- 100 µl 0.5M EDTA pH 8.0 (1 mM final)
- 50 µl 0.5M EGTA pH 8.0 (0.5 mM final)
- 250 µl 10% SDS (Merck) (0.05% final)
- 1% Protease Inhibitor Complete (PIC) (1 tablet, Roche)
1X Incubation buffer (10 ml):
- 100 µl 1M Tris pH 8.0 (10 mM final)
- 300 µl 5M NaCI (150 mM final)
- 20 µl 0.5M EDTA (1 mM final)
- 10 µl 0.5M EGTA (0.5 mM final)
- 150 µl 10% SDS (0.15% final)
- 500 µl 20% Triton (1% final)_
Wash buffer 1 (10 ml):
- 100 µl 1M Tris pH 8.0 (10 mM final)
- 300 µl 5M NaCI (150 mM final)
- 20 µl 0.5M EDTA (1 mM final)
- 10 µl 0.5M EGTA (0.5 mM final)
- 100 µl 10% SDS (0.1 % final)
- 100 µl 10% sodium deoxycholate (DOC) (Sigma) (0.1 % final)
- 500 µl 20% Triton (1% final))
Wash buffer 2 (10 ml):
- 100 µl 1M Tris pH 8.0 (10 mM final)
- 1 ml 5M NaCI (500 mM final)
- 20 µl 0.5M EDTA (1 mM final)
- 10 µl 0.5M EGTA (0.5 mM final)
- 100 µl 10% SDS (0.1 % final)
- 100 µl 10% DOC (Sigma) (0.1 % final)
- 500 µl 20% Triton (1% final)
Wash buffer 3 (10 ml):
- 100 µl 1M Tris pH 8.0 (10 mM final)
- 20 µl 0.5M EDTA (1 mM final)
- 10 µl 0.5M EGTA (0.5 mM final)
- 500 µl 5M LiCI (Sigma) (0.25 M final)
- 500 µl 10% DOC (0.5 % final)
- 500 µl 10% NP-40 (Merck) (0.5 % final)
Wash buffer 4 (10 ml):
- 100 µl 1M Tris pH 8.0 (10 mM final)
- 20 µl 0.5M EDTA (1 mM final)
- 10 µl 0.5M EGTA (0.5 mM final)
Elution Buffer (Prepare fresh):