GSM9893181: RNA-seq of human hepatic stellate LX-2 cells transfected with ATP6AP1-targeting siRNA, replicate 2; Homo sapiens; RNA-Seq
Total RNA extraction from cultured LX-2 hepatic stellate cells was performed using TRIzol reagent following the manufacturer's standard protocol. Briefly, cell pellets were homogenized in TRIzol lysis buffer, followed by chloroform phase separation and isopropanol precipitation. RNA pellets were washed with 75% ethanol, air-dried, and dissolved in RNase-free water. RNA integrity and concentration were assessed via agarose gel electrophoresis and Nanodrop spectrophotometry; RNA integrity number (RIN) ≥7.0 was required for subsequent library construction. mRNA was enriched from qualified total RNA using oligo(dT) magnetic beads to deplete ribosomal RNA. Purified mRNA was fragmented into short fragments under elevated temperature. First-strand cDNA was synthesized with random hexamer primers and reverse transcriptase, followed by second-strand cDNA synthesis using DNA polymerase I. End repair, A-tailing, and adapter ligation were sequentially conducted. Fragments with suitable insert sizes were purified, enriched by PCR amplification, and quantified for paired-end RNA-seq library sequencing.