Heat shock factor 5 is indispensable for pachynema progression and LINE1 silencing during spermatogenesis in mice [RNA-seq]

Single-cell suspensions were prepared by enzymatic digestion of seminiferous tubules with collagenase and trypsin. Cells were loaded onto a 4%–2%–0.5% BSA gradient and allowed to sediment for 1 hour and 45 minutes at 4°C. Fractions were collected, and pachytene spermatocytes were identified by SYCP3, γH2AX, and DAPI staining under microscopy.

Enriched pachytene cells were pooled and processed for RNA-Seq.