title: GSM8035761: HeLa, untreated, rep2; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from cultured cells using TRIzol reagent. mRNA was isolated from total RNA using magnetic beads with poly-T oligo attached. Fragmentation was conducted by subjecting the RNA to elevated temperature in the presence of divalent cations and the First Strand Synthesis Reaction Buffer. First strand cDNA synthesis was performed using M-MuLV Reverse Transcriptase (RNase H-) and random hexamer primers. Subsequently, second strand cDNA synthesis was carried out using DNA Polymerase I and RNase H. Following adenylation of the 3' ends of DNA fragments, adaptors with hairpin loop structures were ligated to facilitate hybridization. The library was purified using the AMPure XP system. PCR was then performed using Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. Finally, the PCR products were purified.