GSM9921176: Vector_3; Homo sapiens; RNA-Seq
RNA was extracted from cell culture using TRIzol reagent. Briefly, cell culture 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 is enriched for small RNAs, followed by 3' and 5' adapter ligation. Reverse transcription generates cDNA, which is then PCR amplified with indexed primers. The final library is size-selected (typically ~150 bp) and validated by Bioanalyzer before sequencing on an Illumina flow cell.