Chromatin Immunoprecipitation and Real-Time PCR Protocol

Real-time PCR was conducted as previously described ^{60 (link), 61 (link)}. ChIP experiments were performed using a SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology, 9003) per the manufacturer’s instructions. After chromatin digestion and sonication, antibodies against target proteins as well as a control antibody were used for ChIP. The enriched promoter fragments captured by antibodies were examined using real-time PCR. The signal relative to input was evaluated using a formula from the manufacturer’s protocol as follows: percent input = 2% × 2^{(C[T]2% input sample − C[T]IP sample)}, where C[T] = threshold cycle of PCR reaction. The sequences of primers used for real-time PCR and ChIP-qPCR are listed in Table S4. The primary antibodies used for ChIP-qPCR were: H2Aub (Cell Signaling Technology, 8240), RNA pol II CTD (Cell Signaling Technology, 2629), RNA pol II CTD phospho Ser5 antibody (Active Motif, 61085), RNA pol II CTD phospho Ser2 antibody (Active Motif, 61083), FOXK1 (Abcam, ab18196 ), FOXK2 (Bethyl Laboratories, A301–730A), HCFC1 (Bethyl Laboratories, A301–399A), OGT (Cell Signaling Technology, 5368), KDM1B (Abcam, ab193080), ASXL1 (Santa Cruz, sc-293204).