9. Resuspend the pellet in 3 mL immunoprecipitation buffer.
10. Place the sample in the lysing matrix B 2 mL tubes.
11. Sonicate the samples using the Bioruptor for 45 s, frequency
6.5, this will shear the DNA into small fragments. To obtain
fragments of 300–700 bp, repeat Bioruptor for 35 cycles, 30 s
ON/OFF.
12. Centrifuge at 13,000 Â g for 15 min at 4
C, this will remove
cell debris.
13. Take 14 μL of each sample and add 4 μL 5Â elution buffer and
1 μL RNase, leave at room temperature (RT) for 30 min.
14. Freeze the remainder of each sample at À20
C.
15. Add 2 μL proteinase K to the 14 μL samples from step 13 and
incubate at 42
C for 2 h. De-crosslink at 65
C for 6 h (can be
incubated in a PCR machine). Run on 1.2% agarose gel at
100 V for 35–45 min (see Note 6).
16. Take the remainder (~2986 μL from step 14) and add 25 μL of
protein G magnetic beads to preclear the chromatin (see
Note 7).
17. Incubate the chromatin and beads at 4
C for 1.5 h in a rotator
at 20 rpm.
18. Immobilize the beads using a magnetic rack and transfer the
liquid (cleared chromatin) to a fresh 1.5 mL microcentrifuge
tube (see Note 8).
19. Take 100 μL of the pre-cleared chromatin from step 18 (input
control sample) and store at À20
C.
20. To the remaining chromatin material (~2886 μL), add the
antibody for the protein of interest and incubate for 2.5 h or
O/N at 4
C in a rotator at 20 rpm.
21. Add 30 μL of magnetic beads to the chromatin from step 20
and incubate for 1.5 h at 4
C in a rotator at 20 rpm.
22. Immobilize the beads using a magnetic rack, wait for 2 min,
and remove the liquid (keep this separately at À20
C, see
Note 9).
23. Resuspend the beads by gentle pipetting (do not vortex) in
750 μL ice-cold IP buffer (see Subheading 2.2, item 11).
24. Transfer the resuspended beads to a new nonstick 1.5 mL
microcentrifuge tube.
25. Incubate the resuspended beads in IP buffer at 4
C for 10 min
in a rotator at 20 rpm.
26. Immobilize the beads using a magnetic rack, incubate for
2 min, and discard the IP buffer.
66
Heena Jagatia and Daire Cantillon
10. Place the sample in the lysing matrix B 2 mL tubes.
11. Sonicate the samples using the Bioruptor for 45 s, frequency
6.5, this will shear the DNA into small fragments. To obtain
fragments of 300–700 bp, repeat Bioruptor for 35 cycles, 30 s
ON/OFF.
12. Centrifuge at 13,000 Â g for 15 min at 4
C, this will remove
cell debris.
13. Take 14 μL of each sample and add 4 μL 5Â elution buffer and
1 μL RNase, leave at room temperature (RT) for 30 min.
14. Freeze the remainder of each sample at À20
C.
15. Add 2 μL proteinase K to the 14 μL samples from step 13 and
incubate at 42
C for 2 h. De-crosslink at 65
C for 6 h (can be
incubated in a PCR machine). Run on 1.2% agarose gel at
100 V for 35–45 min (see Note 6).
16. Take the remainder (~2986 μL from step 14) and add 25 μL of
protein G magnetic beads to preclear the chromatin (see
Note 7).
17. Incubate the chromatin and beads at 4
C for 1.5 h in a rotator
at 20 rpm.
18. Immobilize the beads using a magnetic rack and transfer the
liquid (cleared chromatin) to a fresh 1.5 mL microcentrifuge
tube (see Note 8).
19. Take 100 μL of the pre-cleared chromatin from step 18 (input
control sample) and store at À20
C.
20. To the remaining chromatin material (~2886 μL), add the
antibody for the protein of interest and incubate for 2.5 h or
O/N at 4
C in a rotator at 20 rpm.
21. Add 30 μL of magnetic beads to the chromatin from step 20
and incubate for 1.5 h at 4
C in a rotator at 20 rpm.
22. Immobilize the beads using a magnetic rack, wait for 2 min,
and remove the liquid (keep this separately at À20
C, see
Note 9).
23. Resuspend the beads by gentle pipetting (do not vortex) in
750 μL ice-cold IP buffer (see Subheading 2.2, item 11).
24. Transfer the resuspended beads to a new nonstick 1.5 mL
microcentrifuge tube.
25. Incubate the resuspended beads in IP buffer at 4
C for 10 min
in a rotator at 20 rpm.
26. Immobilize the beads using a magnetic rack, incubate for
2 min, and discard the IP buffer.
66
Heena Jagatia and Daire Cantillon