title: RNA-seq of Sus scrofa:7d male testis Sertoli cells
design: RNA-seq Library Preparation: RNA-seq libraries were constructed by enriching polyadenylated mRNA from total RNA using oligo(dT) magnetic beads, followed by in vitro fragmentation to ~300 bp fragments via divalent cation hydrolysis. This fragment size optimizes sequencing efficiency: shorter fragments increase adapter dimer formation, reducing usable data yield, while longer fragments impede cluster generation during flowcell loading. First-strand cDNA was synthesized from fragmented RNA using random hexamer priming and reverse transcriptase, with second-strand synthesis subsequently performed.Amplification via PCR enriched adapter-ligated fragments, followed by size selection for ~450 bp libraries. Library quality was assessed using an Agilent 2100 Bioanalyzer, with quantification via qPCR determining effective library concentrations. Indexed libraries were proportionally pooled based on these concentrations and target sequencing depth. Pooled libraries underwent dilution to 2 nM and alkaline denaturation to generate single-stranded DNA.Following RNA extraction, purification, and library construction, paired-end sequencing (PE152) was performed on the Illumina platform using standard NGS protocols.