RNA-seq library construction

Preleptotene, leptotene, and zygotene spermatocytes were purified and assessed from 10-day-old unsynchronized mice, 16-day-old synchronized mice, and 2-week-old unsynchronized mice, respectively, using the same stringent gating strategies as for constructing Hi-C libraries. The viability and purity of isolated cells are summarized in Supplementary Table ². For each stage, two independent biological samples were isolated and used for RNA-seq library construction. Total RNA was extracted from ~50,000 purified spermatocytes by the RNeasy Micro Kit (Qiagen, 74004) according to the manufacturer’s instructions. The cDNA was synthesized and amplified using the Single Cell Full-Length mRNA-Amplification Kit (Vazyme, N712). The RNA-seq libraries were constructed from the amplified cDNA using the TruePrep DNA Library Prep Kit V2 for Illumina® (Vazyme, TD503). The PCR and size selection of RNA-seq libraries were conducted as described in Hi-C library construction. The RNA-seq libraries were sequenced on Illumina Nova Seq 6000 platform at PE150 mode.