Anti-dimethyl-Histone H3 (Lys27) Antibody

Protocol tips

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	ChIP assays were performed using the EpiQuik™ Tissue Chromatin Immunoprecipitation kit (Epigentek Group Inc.), as previously reported [13-15]. Briefly, small pieces of normal and PCOS ovarian tissues (1-2 mm3) were crosslinked with 1% formaldehyde. Cross-linking was terminated using 1.25 M glycine. The tissues and cells were added to homogenizing buffer, triturated, disaggregated, and centrifuged at 1000 g for 5 min at 4°C. After the removal of supernatants, protease inhibitors were added and the disaggregated tissue pellet was resuspended. Chromatin was sheared by sonication. Immunoprecipitation was performed at room temperature for 90 min.

Protocol tips

ChIP assays were performed using the EpiQuik™ Tissue Chromatin Immunoprecipitation kit (Epigentek Group Inc.), as previously reported [13-15]. Briefly, small pieces of normal and PCOS ovarian tissues (1-2 mm3) were crosslinked with 1% formaldehyde. Cross-linking was terminated using 1.25 M glycine. The tissues and cells were added to homogenizing buffer, triturated, disaggregated, and centrifuged at 1000 g for 5 min at 4°C. After the removal of supernatants, protease inhibitors were added and the disaggregated tissue pellet was resuspended. Chromatin was sheared by sonication. Immunoprecipitation was performed at room temperature for 90 min.

Publication protocol

ChIP assays were performed using the EpiQuik™ Tissue Chromatin Immunoprecipitation kit (Epigentek Group Inc.), as previously reported [13-15]. Briefly, small pieces of normal and PCOS ovarian tissues (1-2 mm3) were crosslinked with 1% formaldehyde. Cross-linking was terminated using 1.25 M glycine. The tissues and cells were added to homogenizing buffer, triturated, disaggregated, and centrifuged at 1000 g for 5 min at 4°C. After the removal of supernatants, protease inhibitors were added and the disaggregated tissue pellet was resuspended. Chromatin was sheared by sonication. Immunoprecipitation was performed at room temperature for 90 min. The specific antibodies (H3K9ac, ab4441; H3K18ac, ab1191; H3K4me1, ab8895; H3K4me2, ab7766; H3K4me3, ab8580; H3K36me3, ab9050; H3K79me, ab2886; H3K9me, ab9045; H3K9me3, ab8898) and (H3K27ac, 07-360; H3K9me2, 07-441; H3K27me, 07-448; H3K27me2, 07-452) for ChIP were purchased from Abcam and Millipore, respectively. Crosslinking was reversed at 65°C for 90 min. Finally, genomic DNA was eluted for PCR analysis. The specific primer sequences for ChIP are listed in Table 1. The thermocycle was 94°C for 2 min, then 30 cycles of 45 s at 94°C, 45 s at 56°C and 45 s at 72°C.

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