ChIP and qPCR Analysis of Epigenetic Regulators

ChIP assay and qPCR analysis were performed as described previously (21 (link)). Primers used in these assays were as follows: for MYOG, 5′-GGAGAAAGAAGGGGAATCACA-3′ and 5′-GATAAATATAGCCAACGCCACA-3′; for GAPDH, 5′-CGCTCTCTGCTCCTCC-3′ and 5′-TTTCTCTCCGCCCGTCCAC-3′; for ADAM control, 5′-ACAGGAGCATGCACTCTTCA-3′ and 5′-GGCAATGTTCTGCTGCAA-3′; for the ADAM peak, 5′-CTTCATGGCTACAGACTCTTGG-3′ and 5′-CCTATGTCTCGCTTCCTGCT-3′; for COBLL1 control, 5′-CCCTCCAGTATACCCCAGCT-3′ and 5′-ACCCCTTCTCTTTACTTGGCC-3′; for the COBLL1 peak, 5′-CTGAGTAACAAGAGCGAAAGAG-3′ and 5′-ATCAGATGTGTTATGACTAACAGC-3′; and for the COBLL1 TSS, 5′-GCCGCCGTCTCTACAAGGTCTA-3′ and 5′-CTACCCAGTAAACCCCACGG-3′. Antibodies used for ChIP experiments were as follows: antibodies against H3K27me3 (07-449; Millipore), H3K4me3 (17-614; Millipore), H3K27Ac (05-1334; Millipore), EBNA3C (ab16128; Abcam), RBPJ (ab25949; Abcam), BMI1 (A301-694A; Bethyl), and SUZ12 (Ab12073; Abcam). Input DNA was 5% of the DNA used for immunoprecipitations and was diluted to 2.5% prior to PCR quantification. Enrichment relative to the input level was calculated using four 5-fold-dilution series, and error bars were calculated as standard deviations for triplicate PCRs for both input and IP samples. All ChIPs shown are representative of at least two independent experiments, each performed on LCLs established by two independent primary B cell infections.