GSM9679233: bmh21_pre-rRNA; Mus musculus; OTHER
Adult mouse testes (C57BL/6, 4-6 weeks) were collected and decapsulated. Single-cell suspension was prepared by enzymatic digestion. Pachytene spermatocytes were isolated by FACS using Hoechst 33342 staining. Purified pachytene spermatocytes were crosslinked with 1% glutaraldehyde for 10-15 min at room temperature with gentle rotation. Crosslinking was quenched by adding 0.125 M glycine (final concentration) and incubating for 5 min at RT. Cells were washed twice with ice-cold PBS and pelleted by centrifugation (800g, 5 min, 4°C).  Cell pellets were resuspended in ice-cold lysis buffer (50 mM Tris-HCl pH 7.0, 10 mM EDTA, 1% SDS, 1× protease inhibitor cocktail, 40 U/mL RNase inhibitor) and incubated on ice for 10 min with occasional vortexing. Chromatin was sheared to 200-600 bp fragments using a Bioruptor. ChIP-enriched DNA (1-10 ng) was used for library preparation using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro. Libraries were amplified by 12-15 cycles of PCR, purified with VAHTS DNA Clean Beads. Quantification was performed by Qubit, followed by pooling for Illumina NovaSeq 6000 sequencing (PE150).