title: GSM5184230: Sertoli Input rep 2 (P7); Mus musculus; ChIP-Seq
library_construction_protocol: Cells were fixed with 1% methanol-free formaldehyde in PBS (Thermo scientific #28906) for five minutes at RT with rotation.  Fixation was stopped by addition of glycine to 0.125M and rotation at RT for 10 min.  After pelleting, cells were washed in PBS and stored at -80o C until use.  Sertoli cell ChIP was performed with about 2.5 million cells and granulosa cell ChIP with about 1 million cells.  Nuclei were prepared from the fixed cells and chromatin was sheared to an average size of 300-400 bp in a Covaris S220 according to the manufacturer's recommendations.  After shearing, the lysate was diluted 1:3 with complete DOC RIPA (37) and the sample was centrifuged at 21,000g for ten minutes at 4o C to pellet any insoluble material.  The supernatant was transferred to a fresh siliconized tube and the ChIP was initiated with the addition of ~1 ug of the relevant antibody and rotated at 4o C overnight.  Samples were then spun at 21,000g for 10 min. to pellet insoluble material and supernatant was mixed with 20 ul Protein A Dynabeads (Invitrogen, 10002D) previously blocked with BSA and yeast tRNA to inhibit non-specific binding.  After incubation at 4o C for 60 min. with rotation to allow association of protein A with the antibody, beads were applied to a magnet and washed sequentially as described previously (37).  After elution and cross link reversal, sequencing libraries were prepared using the Hyper Prep Kit (KAPA Biosystems, KK8502). Whole gonad ChIP-seq was performed essentially the same as for primary cells ChIP-seq except gonads from 2-3 animals were pooled and fixed in 1% paraformaldehyde (Electron Microscopy Sciences #15710) for 10 min. (ESR2 and FOXL2) or fixed in 2 mM disuccinimidyl glutarate (DSG, ThermoFisher Scientific #20593) at RT for 45 min. followed by 1% paraformaldehyde fixation at RT for 15 minutes (SOX9).  After quenching the fix with 0.125M glycine, chromatin was sheared to an average size of 300-500 bp using a tip sonicator.