Methylation sequencing of humo sperm: male sperm cell
For DNA library construction, all DNA samples were subjected to RRBS library preparation using the Acegen Rapid RRBS Library Prep Kit (Acegen, Cat. No. AG0422) according to the manufacturers protocol. In brief, 100 ng of input DNA was digested with MspI. The DNA fragments underwent end repair, A-tailing, and cytosine ligation. Fragments ranging from 150 to 300 base pairs were gel-extracted and subjected to bisulfite treatment using the EZ DNA Methylation Gold Kit (Zymo Research). After bisulfite treatment, these fragments were then PCR-amplified to construct the final DNA libraries. The library quality was assessed using Qubit 2.0, Agilent 2100, and q-PCR for estimating insert size and concentration.After comprehensive DNA library analysis, the fragments were sequenced using the Illumina NovaSeq 6000 system (Illumina, Inc., San Diego, CA, USA), with the resulting raw data being stored in the FASTQ format. Base calling was performed using Illumina Casava 1.8, and data quality was assessed and refined using FastQC and Trimmomatic.