RNA-seq of HK2 cells
For eukaryotic species, leveraging the polyadenylated (polyA) tail structure of mRNA, total RNA was first enriched for mRNA using oligo(dT) beads. The purified mRNA was fragmented with divalent cations, followed by double-stranded cDNA synthesis using fragmented RNA as templates and random oligonucleotide primers. The cDNA was end-repaired, adenylated (dA-tailed), and ligated to sequencing adapters. The ligated products were purified, size-selected to retain desired fragments, PCR-amplified, and purified again to generate the final sequencing library.