GSM9540700: Control_2; Mus musculus; RNA-Seq
Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) according to the manufacturer's instructions. RNA concentration and purity were measured using NanoDrop spectrophotometer. RNA integrity was assessed using Agilent Technologies 2100 Bioanalyzer. Only RNA samples with high integrity (RIN ≥ 7.0) were used for library preparation. Poly(A) RNA sequencing libraries were prepared using Illumina TruSeq Stranded mRNA Library Preparation Kit according to the manufacturer's protocol. Briefly, Poly(A)+ RNA was purified from total RNA using oligo(dT) magnetic beads. Purified RNA was fragmented using divalent cations at elevated temperature. First-strand cDNA synthesis was performed using reverse transcriptase and random primers. Second-strand cDNA synthesis was performed to generate double-stranded cDNA. cDNA fragments underwent end repair, A-tailing, and ligation of sequencing adapters. Adapter-ligated fragments were PCR amplified to enrich the library. Library quality and fragment size distribution were assessed using Agilent Bioanalyzer High Sensitivity DNA chip. Libraries were sequenced using Illumina NovaSeq 6000 platform.