Sperm RNA Extraction and Sequencing

Three biological replicates of fresh-frozen sperm samples and cryopreserved sperm samples were used for RNA extraction, which underwent the same thawing procedure (40 °C water bath for 20 s). To achieve high reproducibility, the sperm samples were homogenized using a PowerLyzer24 instrument (Qiagen, Redwood City, CA, USA). Total RNA was extracted using AllPrep DNA/RNA Kit (Qiagen, Redwood City, CA, USA) following the manufacturer’s instructions. RNA concentrations were measured with the NanoDrop OneC Microvolume Spectrophotometer (Thermo Scientific, Waltham, MA, USA). Evaluation of RNA quality was conducted using the LabChip GX Touch HT (PerkinElmer, Hopkinton, MA, USA). RNA-seq library construction was performed following the procedure described in our previous publication [96 (link)] with 500 ng of total RNA input. The size distributions of cDNA libraries were checked using the TapeStation 4200 D1000 ScreenTape (Agilent Technologies, Santa Clara, CA, USA), and library concentrations were determined by a Qubit 3.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). The libraries were commercially sequenced on the Illumina NovaSeq 6000 platform with a 2 × 150 Paired-End configuration at Novogene (Novogene Corporation Inc., Sacramento, CA, USA).