RNA-Seq of HeLa-S3 UV-exposed, 60mins post-UV
The RNA sample preparations involved using 0.4 g of RNA per sample with the NEBNext UltraTM RNA Library Prep Kit for Illumina (NEB, USA) following the manufacturers guidelines, incorporating index codes for sample attribution. Initially, mRNA purification was executed using poly-T oligo-attached magnetic beads, followed by fragmentation in NEBNext First Strand Synthesis Reaction Buffer (5X) under increased temperature with divalent cations. First strand cDNA synthesis was conducted with random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-), followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. Blunt ends were generated from remaining overhangs through exonuclease/polymerase activities. Adenylation of DNA fragment ends preceded ligation with NEBNext Adaptors featuring a hairpin loop structure for hybridization preparation. Selection of cDNA fragments, preferably 250~300 bp, was achieved via purification using the AMPure XP system (Beckman Coulter, Beverly, USA). Subsequent PCR employed Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer. The resulting PCR products underwent purification with the AMPure XP system, and library quality was assessed using the Agilent Bioanalyzer 2100 system.