Trizol The mRNA was fragmented with fragmentation buffer, and short RNA fragments were used as templates for synthesizing the first strand of cDNA with random hexamer primers. The second strand of cDNA was then synthesized using buffer, dNTPs (dTTP, dATP, dGTP, and dCTP), and DNA polymerase I. The double-stranded cDNA was purified with DNA purification beads, followed by end-repair, A-tailing, and ligation with sequencing adapters. After selecting fragments of the appropriate size using DNA purification beads, the final cDNA library was enriched via PCR.