SWITCHGEAR RECOMMENDED CHROMATIN IP PROTOCOL
Day 1
1. Add formaldehyde to 2e7 cells (approx 5 x 105 cells/ml) to a final concentration of 1% from 18.5% stock
2. Fix at 25°C (room temp) for 10 minutes
3. Stop cross-linking reaction by adding glycine to a final concentration of 0.125 M from a 2.5 M stock
4. Pellet cells by spinning at 2000 rpm for 5 min and wash once with ice cold PBS
5. Resuspend in 6 mL LYSIS BUFFER and mix gently.
6. Collect crude nuclear prep by microcentrifugation at 2000 rpm for 5 min
7. Resuspend in ~1.9 mL RIPA BUFFER and transfer to 2 mL eppy
8. Sonicate on ice at power output 5-6 and continuous, 4 times for 30 sec cooling on ice in between (ave length of fragments 400-600 bp)
9. Microcentrifuge at 14,000 rpm for 15 min @ 4°C and save cleared chromatin prep supernatant, snap freeze in LiN, store at -80 or continue with IP
Day 2: Coupling Antibody
a. Add 50 ul of resuspended magnetic bead slurry to 1.5 ml eppy (Dynal magnetic beads with appropriate secondary antibody)
b. Place tube on magnet, remove supernatant
c. Resuspend beads in 1 ml PBS + 5 mg/ml BSA (prepare fresh)
d. Repeat steps b and c, 3 times to wash
e. Add 1 ml PBS+BSA to beads
f. Add 5 ug of primary antibody (25 uL of 200 ug/ml for polyclonals)
g. Mix on rotator platform overnight at 4 degrees
h. Wash 3X (steps b-c)
i. Resuspend in 100 ul PBS+BSA, add to chromatin prep, incubate @ 4°C overnight
Day 3:
10. Perform all washes and spins at 4°C
11. Pellet on magnet, save supernatant to reverse crosslinks to make mockIP DNA and to run on gel
12. Wash pellet 5X with 1 ml LiCl IP WASH BUFFER with 3 min mixing between washes
13. Wash pellet once with 1 ml TE
14. Resuspend beads in 200 uL IP ELUTION BUFFER
15. Elute immune complexes by incubating in 65°C water bath for 1 hr, shaking every 15 min to resuspend beads
16. Spin 14,000 rpm for 3 min to pellet beads, save supernatant
17. Continue cross-link reversal by incubating in 65°C water bath overnight
18. Also reverse cross-links of mockIP supernatant in 65°C water bath overnight
Day 4:
19. Use 3-4 DNeasy columns to clean up the DNA from all the mockIP supernatant. This will serve as a negative control for downstream analyses.
20. Extract reverse crosslinked ChIP supernatant once with 200 ul of phenol/chloroform/isoamyl alcohol, vortex thoroughly, and separate phase by spinning 14,000 rpm for 3 minutes
21. Save aqueous phase, back extract organic phase once with 50 uL water and pool both aqueous phases
22. Add 750 uL (3X aqueous volume) Qiagen Buffer PM (PCR cleanup kit)
23. Add half (~500 uL) of the solution to Qiagen PCR cleanup column, spin, repeat with other half to bind 1 ml sample on 1 column
24. Wash DNA on column with 750 uL Buffer PE, spin, empty, spin to dry
25. Elute with 100 uL warmed Buffer EB (~55°C), run eluate over same column again for second elution
26. For amplification, speed-vac concentrate to a volume of 14 ul
27. To test with real-time PCR, dilute eluted ChIP DNA 10X and use 10 ul as temp
28. Run extracted mockIP DNA on 1% gel to check fragment size
SOLUTIONS
Lysis buffer:
5mM PIPES pH 8.0
85mM KCl
0.5% NP-4
*Roche Protease Inhibitor Cocktail
RIPA buffer:
1 X PBS
1% NP-40
0.5% Sodium Deoxycholate
0.1% SDS
*Roche Protease Inhibitor Cocktail
IP elution buffer:
1% SDS
0.1 M NaHCO3
LiCl IP WASH BUFFER:
100 mM Tris
500 mM LiCl
1% NP-40
1% Deoxycholate