GSM8675806: C_rep2; Mus musculus; Bisulfite-Seq
The cauda epididymis was dissected from the mice, placed in pre-warmed M2 medium (Sigma, N7167), and gently squeezed to release the caudal fluid into the media. The sperm containing media was incubated at 37°C for 30 minutes and then transferred to a fresh tube through cell filter. Sperm cells were collected by centrifugation at 800xg for 5 minutes, followed by a wash with 1X PBS and centrifugation step. After removing the supernatant, all samples were treated with somatic lysis buffer (SLB: 0.1% SDS, 0.5% Triton-X-100 in DEPC water) on ice for 20 minutes, followed by centrifugation at 1000xg for 5 minutes. The treated sperm was washed twice with 1X PBS and centrifuged at 4000xg for 3 minutes. The sperm DNA was extracted using TIANamp Micro DNA Kit. A total of 100 ng of genomic DNA was fragmented to 350 bp using Hieff® Smearase (YEASEN, 12907ES24). Subsequently, the DNA was bisulfite converted with EpiArt DNA Methylation Bisulfite Kit (Vazyme, EM101-01), and the WGBS library was prepared using EpiArt DNA Methylation Library Kit for Illumina V3 (Vazyme, NE103-01) following the manufacturer's instructions. The libraries were purified using VAHTS DNA Clean Beads (Vazyme, N411-01). In each group, the WGBS libraries of 3 mice sperm were pooled into a sequencing library in equal proportions and then sequenced on the Illumina NovaSeq 6000 in paired-end mode (PE150).