GSM8365383: Sample 1-3_control; Homo sapiens; Bisulfite-Seq
All individuals were required to provide a semen sample by masturbation that was collected after a previous ejaculation that occurred 3-7 days ago. Subsequently, semen samples were frozen at -80℃. Semen samples underwent washing with 10 mL of 1X phosphate-buffered saline, followed by centrifugation and resuspension of the pellet in 10 mL of somatic cell lysis buffer (0.1% SDS, 0.5% Triton X-100 in DEPC H2O; Sigma-Aldrich). Overnight incubation at 4℃ was followed by multiple centrifugations and resuspension of the pellet in 10 mL phosphate-buffered saline. Sperm DNA underwent treatment using the sodium bisulfite-based EZ DNA Methylation-Gold™ Kit, effecting the conversion of unmethylated cytosine (C) to uridine (U) while preserving the methylated cytosine state. Specific primers targeting CpG island sequences were generated using Primer3. High-throughput sequencing was performed on an Illumina Hiseq with a 2×150bp paired-end configuration.