RNA-Seq of Human cancer cell lines MHCC97H
Polyadenylated mRNA was enriched using oligo(dT) magnetic beads (Hieff NGS mRNA Isolation Master Kit, Yeasen). Fragmentation to 200-300 bp was performed via magnesium-based buffer incubation at 94C (Hieff NGS Ultima Dual-mode RNA Library Prep Kit). First-strand cDNA synthesis utilized random hexamer priming with M-MuLV reverse transcriptase, followed by RNase H digestion and second-strand synthesis incorporating dUTP for strand specificity. Double-stranded cDNA underwent end repair, A-tailing, and Illumina/BGI-compatible adapter ligation (Hieff NGS Ultima reagents). Libraries were size-selected (~200 bp) using magnetic beads, treated with UDG to degrade dUTP-marked second strands, and PCR-amplified (ABI 9700). Final libraries were quality-controlled via Qubit fluorometry (2 nM), Agilent 2100 Bioanalyzer (peak 350-450 bp, DV200 >80%), and agarose gel electrophoresis.