GSM9075889: Vector control replicate 3 (PL45); Homo sapiens; RNA-Seq
Total RNA was isolated with TRIzol reagent (Thermo Fisher, Cat #15596018) and purified using the RNeasy Mini Kit (QIAGEN) with on-column DNase I digestion. RNA integrity was assessed on an Agilent 2100 Bioanalyzer; only samples with RIN ≥ 8.0 were advanced Strand-specific, paired-end libraries were prepared from 1 µg high-quality RNA using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (NEB, E7760) with rRNA depletion (NEBNext rRNA Depletion Kit, Human/Mouse/Rat). Fragmentation, first-/second-strand cDNA synthesis, end repair/A-tailing, adapter ligation (NEBNext Multiplex Oligos), size selection (~300 bp), and 12-cycle PCR enrichment were performed per the kit manuals. Libraries were quantified by Qubit and qPCR, pooled equimolarly, and sequenced (2 × 150 bp) on an Illumina NovaSeq 6000 S4 flow cell.