ChIP-qPCR Assay for Histone Marks

Cells in 10cm² dishes were fixed in 1% formaldehyde for 5 minutes and fixation was quenched with addition of glycine to 125 mM for an additional 5 minutes. Cells were harvested by scraping from plates, and washed twice in 1× PBS before storage at −80°C. ChIP was performed as previously described (44 (link)) except that extracts were sonicated six times for 7.5 minutes each round (30 seconds sonication with intermediate incubation of 30 seconds per round) using a Bioruptor (Diagenode). All ChIPs were performed using 150 μg of extract and 2 μg of antibody per sample. 30 μl of Protein G Dynabeads (Invitrogen 100.02D) were used per ChIP. Antibodies included Anti-Histone H3 (Abcam ab1791), Anti-Histone H3 (tri methyl K9) (Abcam ab8898), Anti-Histone H3 (tri methyl K4) (Abcam ab8580), Anti-Histone H3 (tri methyl K27) (Abcam ab6002). Following elution, ChIP DNA was analyzed by standard qPCR methods on a 7900HT Fast-Real-Time PCR (ABI). Primer sequences are available upon request.