ChIP-seq Analysis of Histone Modifications

ChIP analysis was performed as previously described with minor modifications 28 (link). Briefly, cells were fixed with 1% formaldehyde for 15 mins at room temperature, and then quenched with 2.5 M glycine. Cells were lysed on ice for 10 mins in SDS buffer containing protease inhibitors, and then sonicated with a Sonics Vibra Cell^TM (6 mins: pulse 10 sec, interval 10 sec) on ice. The fragmented chromatin samples were centrifuged at 8000 × g at 4 °C for 5 mins and supernatant was collected. The samples were pre-cleared, and then incubated overnight at 4 °C with appropriate antibodies and protein-coated A/G agarose beads (Santa Cruz, CA, USA) with gentle shaking. The immunoprecipitated eluates were reverse cross-linked and recovered through DNA purification for PCR. Anti-H3K4me3 (Abcam, ab1012), anti-H3K9ac (Abcam, ab12179), anti-H3K27me3 (Abcam, ab6002), anti-EZH2 (Cell Signaling, 5246S), and non-immune mouse IgG (Santa Cruz, sc2025) antibodies were used. ChIP-PCR primers are listed in Table 1.