GSM9431454: HCT116_AFZC_02; Homo sapiens; RNA-Seq
Total RNA was extracted from tissues or cells using a standard extraction method. RNA quality control was performed primarily using the Agilent 2100 Bioanalyzer to precisely assess RNA integrity (RIN) and quantity. Only samples passing quality thresholds proceeded to library construction. Detailed metrics are provided in the sample QC report. Libraries were constructed using the standard (non-strand-specific) protocol. Briefly, polyadenylated mRNA was enriched from total RNA using Oligo(dT) magnetic beads. The enriched mRNA was then fragmented using divalent cations under elevated temperature. First-strand cDNA synthesis was performed using random hexamer primers, followed by second-strand cDNA synthesis. The double-stranded cDNA underwent end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification to generate the final sequencing library. Library quality was assessed using a Qubit 2.0 Fluorometer for preliminary quantification, an Agilent 2100 Bioanalyzer for insert size distribution, and qPCR for accurate quantification of effective library concentration. Qualified libraries were pooled and sequenced on the Illumina platform using sequencing-by-synthesis (SBS) chemistry.