RNA-seq of Homo sapiens: AAH hPGCLCs, Rep2
After the RNA sample passes the test, magnetic beads with Oligo(dT) are used to enrich eukaryotic mRNA. Then fragmentation buffer is added to break the mRNA into short fragments. Using the mRNA as a template, six-base random primers (random hexamers) are used to synthesize one-strand cDNA. Then buffer, dNTPs, and DNA polymerase I and RNase H are added to synthesize two-strand cDNA. Double-stranded cDNA was then purified using AMPure XP beads. The purified double-stranded cDNA is first subjected to end repair, A-tailing and ligation of sequencing adapters, and then AMPure XP beads are used for fragment size selection. Finally, PCR amplification was performed, and the PCR product was purified with AMPure XP beads to obtain the final sequencing library.