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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.|
|-It is highly critical that the chromatin is of appropriate size and concentration.|
|-For optimal ChIP results, use 10 μl of antibody and 10 μg of chromatin (approximately 4 x 106 cells) per IP.
-It is important to keep the tissue cold to avoid protein degradation.
-Use fresh formaldehyde that is not past the manufacturer's expiration date.
|-Once in solution, store 1M DTT at -20°C.|