GSM9409381: A4; Homo sapiens; RNA-Seq
RNA was extracted from tissue using TRIzol reagent. Briefly, cells were washed with PBS, lysed in TRIzol, and incubated for 5 min. Chloroform (20% volume) was added, followed by vigorous mixing and centrifugation (12,000 ×g, 15 min, 4°C) to separate phases. The aqueous layer was collected, mixed with isopropanol, and centrifuged (12,000 ×g, 10 min) to precipitate RNA. The pellet was washed twice with 75% ethanol, air-dried, and dissolved in RNase-free water. RNA concentration and integrity (RIN >7) were assessed using Nanodrop and Bioanalyzer. Samples were stored at -80°C to prevent degradation. This method ensures high-quality RNA for transcriptomic analyses such as RNA-seq or qPCR. Total RNA (1 µg, RIN >7) was enriched for mRNA using poly(A) selection (for eukaryotes) or rRNA depletion (for prokaryotes). The RNA was fragmented (∼200–300 nt) and reverse-transcribed into cDNA using random hexamers and SuperScript IV. Second-strand synthesis was performed with dUTP incorporation (strand-specific libraries). The cDNA was end-repaired, A-tailed, and ligated to Illumina-compatible adapters. Post-ligation cleanup (AMPure XP beads) was followed by size selection (200–500 bp) and uracil digestion (for strand specificity). Library amplification (8–12 cycles) used indexed primers, and final quantification (Qubit) and quality control (Bioanalyzer/TapeStation) ensured optimal fragment size (∼300 bp) and concentration (>10 nM). Libraries were pooled and sequenced (Illumina platforms, 150 bp paired-end).