GSM9677563: A549 SA_2; Homo sapiens; RNA-Seq
Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. RNA concentration and purity were assessed using a NanoDrop One/OneC spectrophotometer (Thermo Fisher Scientific) prior to RNA sequencing. Messenger RNA (mRNA) was isolated from total RNA using oligo(dT)-conjugated magnetic beads. Following fragmentation, first-strand cDNA synthesis was performed using random hexamer primers. Second-strand synthesis incorporated dUTP to generate strand-specific (directional) libraries. Libraries were prepared through end repair, A-tailing, adapter ligation, size selection, PCR amplification, and purification, including a USER enzyme digestion step prior to amplification for strand specificity. Library concentration was measured using a Qubit fluorometer, and library quality and fragment size distribution were assessed using a Bioanalyzer. Libraries were pooled equimolarly and sequenced on an Illumina platform