VP-1
The library preparation started with total RNA, from which polyA-tailed mRNA was enriched using Oligo(dT) magnetic beads. The purified mRNA was then randomly fragmented in Fragmentation Buffer containing divalent cations. Using the fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA was synthesized in an M-MuLV reverse transcriptase system. Subsequently, the RNA strand was degraded by RNaseH, and the second cDNA strand was synthesized using DNA polymerase I with dNTPs as substrates.The double-stranded cDNA was purified, followed by end repair, A-tailing, and adapter ligation. cDNA fragments of approximately 370420 bp were selected using AMPure XP beads, PCR-amplified, and further purified with AMPure XP beads to obtain the final library.