ChIP-seq

1. Fix 10 million cells with a final concentration of 1% formaldehyde for 10 minutes at room temperature.
2. Quench the crosslinking with 125 mM glycine for 5 minutes.
3. Wash the cells with PBS, and collect the cell pellets in 15 ml Falcon. 
4. Resuspend the cell pellets with 700 µl lysis buffer and keep on ice for 10 minutes. 
5. Sonicate the DNA by using Biorupter (30 sec on/off, 4 cycles). 
6. Dilute the sample 1:10 with dilution buffer, and pre-clear the sample with 100 µl Affi-Prep protein A beads (pre-absorb the beads with 0.1 mg/ml BSA overnight and wash 2 times with dilution buffer before use), incubate for 1 h at 4 °C with rotation. 
7. Transfer the supernatant to a new 15 ml Falcon tube, add 12 µg antibody overnight with rotation.
8. Add 100 µl Affi-Prep protein A beads (wash 2 times with dilution buffer before use), incubate 3 hours with rotation.
9. Wash the beads 2 times with wash buffer 1, 2, 3 and TE.
10. Elute 2 times with 200 µl elution buffer for 20 minutes at 65 °C, pool the eluates.
11. Add 25 µl RNase-A and proteinase K, incubate at 37 °C for 1h, then 65°C overnight.
12. Add 1 µl glycogen and 45 µl (1/10 vol) sodium acetate (3 M, pH 5.2), extract with 600 µl of phenol/chloroform/isoamylalcohol, and once more with 600 µl of chloroform. 
13. Precipitate the DNA with 2.5 vol. 100% EtOH overnight at -20°C.
14. Wash with 1 ml 70 % EtOH.
15. Remove supernatant, let the pellet dry for 10 minutes at room temperature. 
16. Dissolve DNA in 100 µl H2O.

Lysis buffer:

50 mM Tris-HCl pH 8.0

10 mM EDTA pH 8.0

1 % SDS

1 mM PMSF

Dilution buffer:

20 mM Tris-HCl pH 8.0

2 mM EDTA pH 8.0

1 % Triton X-100

150 mM NaCl

1 mM PMSF

 

Wash buffer 1:

20 mM Tris-HCl pH 8.0

2 mM EDTA pH 8.0

1% Triton X-100

150 mM NaCl

0.1% SDS

1 mM PMSF

Wash buffer 2:

20 mM Tris-HCl pH 8.0

2 mM EDTA pH 8.0

1% Triton X-100

500 mM NaCl

0.1% SDS

1 mM PMSF

Wash buffer 3:

10 mM Tris-HCl pH 8.0

1 mM EDTA pH 8.0

250 mM LiCl

0.5% NP-40

0.5% deoxycholate

TE buffer:

10 mM Tris-HCl pH 8.0

1 mM EDTA pH 8.0

Elution buffer: 

25 mM Tris-HCl pH 7.5

5 mM EDTA pH 8.0

0.5% SDS