GSM9423282: U251-NC-2; Homo sapiens; RNA-Seq
Total RNA was treated with RQ1 DNase (Promega) to remove genomic DNA contamination. RNA concentration and purity were assessed by UV spectrophotometry (A260/A280, Bio-Rad SmartSpec Plus), and RNA integrity was verified by 1.5% agarose gel electrophoresis. For each sample, 1 μg of total RNA was used for library preparation. Polyadenylated mRNA was enriched using VAHTS mRNA Capture Beads (Vazyme, N401), followed by fragmentation and first- and second-strand cDNA synthesis with dUTP incorporation during second-strand synthesis to retain strand specificity. End repair, A-tailing, and adaptor ligation (Roche, KK8726) were performed, and ligated fragments (300–500 bp) were size-selected, PCR amplified, purified, and quantified. The final libraries were stored at -80°C prior to sequencing. Strand-specific libraries were generated using the KAPA Stranded mRNA-Seq Kit for Illumina® Platforms (KK8544), following the manufacturer's protocol. Sequencing was performed on an Illumina NovaSeq 6000 system to generate 150 bp paired-end reads.