GSM9433347: Kidney, db/m control, rep1; Mus musculus; RNA-Seq
Total RNA was extracted from mouse kidney tissues using a standard RNA extraction method. Briefly, tissues were homogenized in TRIzol reagent, followed by phase separation with chloroform. RNA was precipitated with isopropanol, washed with 75% ethanol, and dissolved in RNase-free water. RNA quality and integrity were assessed using an Agilent 2100 Bioanalyzer with RNA Nano chips. Only RNA samples with RNA Integrity Number (RIN) ≥ 7.0 and clear 28S/18S ribosomal RNA ratios were used for subsequent library construction. mRNA was enriched from total RNA using Oligo(dT) magnetic beads. The enriched mRNA was fragmented using divalent cations under elevated temperature in fragmentation buffer. First-strand cDNA synthesis was performed using random hexamer primers, followed by second-strand cDNA synthesis. For strand-specific libraries, dUTP was incorporated during second-strand synthesis instead of dTTP. The double-stranded cDNA fragments underwent end repair, adenylation of 3' ends, and ligation of Illumina sequencing adapters. Fragments of desired size were selected using AMPure XP beads, followed by PCR amplification to enrich adapter-ligated fragments. Library quality was assessed using Qubit 2.0 Fluorometer for quantification and Agilent 2100 Bioanalyzer for insert size distribution. Libraries with insert sizes matching expectations were quantified by qRT-PCR to determine accurate concentration (requiring > 1.5 nM). Qualified libraries were pooled in equimolar ratios based on effective concentration and sequencing depth requirements for Illumina sequencing.