title: RNA-seq of sus scrofa: male piglet testes
design: RNA purification, reverse transcription, library construction, and sequencing were conducted at Shanghai Majorbio Bio-pharm Biotechnology Co., Ltd. (Shanghai, China), in accordance with the manufacturer's instructions (Illumina, San Diego, America). The RNA-seq transcriptome library was prepared following the Illumina Stranded mRNA Prep protocol, using 1 g of total RNA and ligation reagents from Illumina (San Diego, America). The messenger RNA was isolated using the polyA selection method with oligo(dT) beads and then fragmented using a fragmentation buffer. Secondly, double-stranded cDNA was synthesized using a SuperScript Double-Stranded cDNA Synthesis Kit (ThermoFisher, Shanghai, China) with random hexamer primers (Illumina, San Diego, America). The synthesized cDNA was then subjected to end-repair, phosphorylation, and addition of 'A' bases following Illumina's library construction protocol. Libraries were chosen based on cDNA target fragments of 300 bp on a 2% Low Range Ultra Agarose gel. Subsequently, PCR amplification was carried out using Phusion DNA polymerase for 15 cycles. After quantification with the Qubit 4.0, the paired-end RNA-seq sequencing library was sequenced using the NovaSeq Xplus sequencer with a read length of 2  155 bp.