2.6 Chromatin immunoprecipitation (ChIP) assay
Homogenates of NCI-H295R cells and fetal adrenal were sheared using digestion/nuclei permeabilization buffer containing MNase at 37°C. 1% formaldehyde was added directly to the homogenate of testicular tissue or scraped cells for 15 min at 37°C to cross-link DNA and its associated proteins. Glycine (0.125 M final concentration) was added to terminate the reaction for 8 min. The lysates were then sonicated to shear the DNA to a size of 200-800 bp. After sonication, the samples were collected by centrifugation and were diluted with ChIP dilution buffer. The supernatant was used for each ChIP reaction, supplemented with ChIP dilution buffer and anti H3K27(1:50 dilution, ABclonal, A7253) or goat anti-rabbit IgG (1:50 dilution, ABclonal, AC005). Then, the DNA-protein-antibody complexes were captured with BSA-treated protein G agarose beads (EMD Millipore, Billerica, MA, USA). After incubation for 6 h, beads were washed, and DNA-protein-antibody complexes were eluted, proteins were digested with proteinase K (EO0491, Beyotime Biotechnology, Nanjing, China) and ChIP-DNA was eluted using a DNA purification kits (Tiangen Biotech Co., Beijing, China). DNA from each input sample was extracted in parallel. For quantification by qPCR, ChIP-DNA was diluted and run using the SYBR Green PCR kit with specific primers listed in TableS2. All primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).