GSM9177974: Sperm total RNA, Duroc boar 4, 7 months, pubertal; Sus scrofa; RNA-Seq
Fresh semen containing 200 million sperm/ml was centrifuged at 600×g for 10 min at RT. Sperm pellets were washed with PBS and subjected to somatic cell lysis using 1 mL somatic cell lysis buffer (0.1% SDS, 0.5% Triton X-100) for 30 min on ice, then washed at 200×g for 15 min at 4°C. Sperm were washed twice with PBS (600×g, 10 min, 4°C) and snap-frozen. RNA extraction: sperm lysed with 1 mL Qiazol and 120 µL TCEP, vortexed 5 min, incubated at 56°C for 60 min. RNA isolated using miRNeasy Mini Kit (Qiagen) with on-column DNase digestion, eluted in 40 µL RNase-free water. Total RNA-seq: Using 4 µL RNA (8.8-61.2 ng), rRNA depletion performed with QiaSeq FastSelect HMR reagents following manufacturer's protocol for partially degraded RNA (RIN 2-6). Libraries prepared with NEBNext Ultra II Directional Library Prep Kit from step 4.2, adapters diluted 1:25, 16 PCR cycles amplification. miRNA-seq: Libraries prepared using QiaSeq miRNA library kit with 5.5-38.3 ng RNA input, adapters diluted per manufacturer recommendations, 18 PCR cycles amplification. All samples indexed with unique adapters. miRNA-seq: Libraries prepared using QiaSeq miRNA library kit (Qiagen) with 5.5-38.3 ng total RNA input. Adapters diluted according to manufacturer recommendations based on RNA input amount. Libraries amplified with 18 PCR cycles. Samples indexed with unique adapters to enable multiplexing on single sequencing run.