RNA-Seq of PK15 cells
Total RNA was enriched for polyadenylated mRNA using Oligo(dT) magnetic beads. The isolated mRNA was fragmented to ~300 bp fragments via ionic shearing. This size represents an optimal compromise: shorter fragments increase adapter sequence dominance (reducing usable data), while longer fragments impede efficient cluster generation during sequencing. First-strand cDNA synthesis was performed using random hexamer primers and reverse transcriptase, followed by second-strand cDNA synthesis. The resulting double-stranded cDNA libraries were PCR-amplified and size-selected to ~450 bp. Library quality and concentration were assessed using an Agilent Bioanalyzer and quantitative methods. Libraries were then multiplexed by pooling equimolar amounts based on effective concentration and desired sequencing depth, using unique index sequences for sample identification. The pooled library was diluted to 2 nM and denatured to single strands.