ChIP Assay[IP]

Procedure for 2X105 cells

1. Spin down cells in eppendorf tube (1700 rpm, 5’, 4°C) and discard SN
2. Crosslinking: add 200 ul Formaldehyde (1% freshly diluted in PBS1X)
3. 37°C 10 min
4. Add 1.3 ml cold PBS, 15 ul 100 mM PMSF, 1.5 ul 1 mg/ml aprotinin
5. Spin (2300 rpm, 5 min, 4°C plus quick spin max speed 3 sec) and discard SN
6. Add 200 ul of warmed up SDS lysis buffer, 2 ul PMSF, 0.2 ul aprotinin
7. Leave 10 min on ice
8. Sonicate for about 1 min 30 sec, power level 3 (to be optimised by the user)
9. Spin (12 000 rpm, 10 min, 4°C) and transfer SN to 2ml eppendorf tube
10. Add 1.8 ml dilution buffer, 18 ul PMSF, 1.8 ul aprotinin
11. Keep 100 ul of this solution as Input and to the rest add 40 ul of ssDNA/protA beads (well mixed; with cut yellow tip)
12. Rock 30 min or more, 4°C
13. Spin 1 min, 4000 rpm, 4°C and transfer supernatant to new eppendorf tube + half of the vol of antibodies suggested for 106 cells
14. Rock overnight 4°C
15. Next morning: add 40 ul ssDNA/protA beads and rock for 1 hour at 4°C
16. Spin at 3000 rpm 1 min (not more, to avoid irreversible packaging of beads) 4°C, 1min
17. Add 500 ul Low-Salt Wash Buffer (kit) to beads and rock 3-5 min, 4°C
18. Spin at 4000 rpm 1 min. (keep this SN to put on gel to control molecular weight of sonicated chromatin) Repeat 17-18 four more times with : a) High-salt wash buffer, b) LiCl, c) TE, d) TE
19. Add 250 ul freshly made Elution buffer to beads (EB = 110ul NaHCO3; 935ul H2O, 55ul 20% SDS) and rock for 15 min at Room Temperature
20. Spin at 3000 rpm 1 min; retrieve supernatant KEEP IT!
21. Repeat 19-20 and pool supernatants (this is ChIP sample)
22. Add 20 ul 5M NaCl; bring Input sample and SN of step 18 to 500 ul with H2O and add 20 ul 5M NaCl
23. Leave at 65°C for 4 hours to reverse crosslink
24. Proteinase K digestion: 10 ul 0.5M EDTA, 20 ul 1M Tris pH 6.5, 2 umg/ml Prot K
25. Leave at 55°C for 1 hr
26. Phenol:Chloroform extraction
27. Precipitation with 0.5-2 ul 10 mg/ml tRNA and 2-2.5 vol 100% EtOH
28. Leave at –20°C ON
29. Centrifuge and resuspend in approx. 30 ul H2O