GSM9511248: 12202; Homo sapiens; RNA-Seq
Whole blood RNA was extracted from PAXgene blood RNA tubes. Briefly, frozen tubes were thawed at room temperature and centrifuged according to the manufacturer's protocol. The resulting pellet was then washed with nuclease free water, resuspended in sterile PBS, and split in half. RNA was extracted from one half of the suspension using the QIAsymphony PAXgene Blood RNA Kit (Qiagen). The other half of the suspension was used for DNA extraction. RNA concentration and integrity were assessed using the Ribogreen Assay (Thermo) and a Fragment Analyzer (Agilent), respectively. Polyadenylated RNA was enriched using NEBNext Magnetic Oligo(dT)25 Beads and prepared into RNA-seq libraries using the NEBNext mRNA Library Prep Reagent Set for Illumina (New England BioLabs, Ipswich, MA, USA). Unique dual-index barcodes were incorporated as specified per manufacturer's protocol. Library concentration was measured using the Quant-iT PicoGreen dsDNA Reagent (Thermo), and library size distribution and quality were checked using a DNA chip on Caliper Gx Touch (Revity). Library quantification was further refined by qPCR using the KAPA Library Quantificaiton Kit (Roche). Paired-end sequencing (2 x 100 bp, 100 million reads per sample) was carried out on an Illumina NovaSeq 6000 platform (Illumina) following manufacturer's recommended protocols.