GSM8970702: Vector_2; Homo sapiens; RNA-Seq
Total RNA was isolated via the Trizol method (1) mRNA was isolated using Oligo(dT)-attached magnetic beads. (2) The isolated mRNA was subsequently fragmented randomly in a fragmentation buffer. (3) First-strand cDNA synthesis was performed using the fragmented mRNA as a template and random hexamers as primers. This was followed by second-strand cDNA synthesis, which involved the addition of PCR buffer, dNTPs, RNase H, and DNA polymerase I. The cDNA was then purified using AMPure XP beads. (4) The resulting double-stranded cDNA underwent end repair, with the addition of adenosine at the ends and ligation to adapters. AMPure XP beads were utilized to select fragments within the size range of 300-400 base pairs. (5) The cDNA library was constructed through several rounds of PCR amplification of the cDNA fragments obtained in step 4.