GSM9666981: hTERT977_Rep1_LPS_C; Homo sapiens; RNA-Seq
The cell pellet was lysed, followed by RNA extraction using ExtractRNA (Eurogen, Russia) and the phenol-chloroform method. It was then treated with DNase (NEB, US) according to the manufacturer's protocol. RNA was precipitated with sodium acetate/ethanol and resuspended in RNase-free water. RNA concentration was measured on a Qubit 4 (Thermo Fisher Scientific, USA), and quality was assessed on an Agilent 2100 Bioanalyzer (RNA 6000 Nano; Agilent Technologies, USA). Only samples with RIN > 7 were used for library preparation. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB, USA) with poly(A)-mRNA isolation (NEBNext Poly(A) mRNA Magnetic Isolation Module). Following mRNA fragmentation, double-stranded cDNA synthesis, end processing, A-tailing, and adapter ligation were performed. Enrichment was carried out by PCR using unique dual-indexed primers (NEBNext Multiplex Oligos, UK). Library quality was assessed on an Agilent 2100 Bioanalyzer (DNA 1000).