Chromatin Immunoprecipitation Assay Protocol

ChIP assay were performed as previously described [67 (link)]. Briefly, dissected ovaries were fixed in 1.8% formaldehyde at room temperature for 10 min. Chromatin was sonicated and used for immunoprecipitation with anti-trimethyl-Histone H3 Lys9 (ab8898; Abcam), or anti-dimethyl-Histone H3 Lys4 (ab7766; Abcam) antibodies. DNA precipitates were amplified by real-time quantitative PCR. PCR product levels were normalized to input and expressed relative to a positive control gene (the 1360-element for the immunoprecipitation with the anti-H3K9me3 antibody and Rpl15 for the immunoprecipitation with the anti-H3K4me2 antibody). The relative DNA levels were calculated using the following formula: E(target)^CtIP *E(ref)^CtInput / E(ref)^CtIP * E(target)^CtInput, where E is the efficiency of each primer pair and ref the positive control. Primers are listed in S3 Table.