RNA-Seq of Homo sapiens Rhabdomyosarcoma
After the samples are qualified, the magnetic beads with Oligo (dT) are used to enrich the eukaryotic mRNA by A-T complementary pairing with the polyA tail of the mRNA. Then fragmentation buffer are added to break the mRNA into short fragments, and one-strand cDNA is synthesized with six-base random primers (random hexamers), using mRNA as a template, followed by two-strand cDNA synthesis though adding buffer, dNTPs and DNA polymerase I. Next, the double-stranded cDNA is purified by AMPure XP beads. The purified double-stranded cDNA is then repaired, A-tailed and ligated with a sequencing adapter, then AMPure XP beads are used for fragment size selection, and finally PCR enrichment is performed to obtain the final cDNA library.