GSM8907876: MR2; Mus musculus; RNA-Seq
Total RNA was isolated from mouse kidney tissues using the Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol. RNA integrity and quality were assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and verified by RNase-free agarose gel electrophoresis. Eukaryotic mRNA was subsequently enriched from the total RNA using Oligo (dT) magnetic beads, while prokaryotic rRNA was depleted using the Ribo-Zero™ Magnetic Kit (Epicentre, Madison, WI, USA). The enriched mRNA was fragmented into short segments using a fragmentation buffer and reverse-transcribed into first-strand cDNA using random primers. Second-strand cDNA synthesis was performed using DNA polymerase I, RNase H, dNTPs, and reaction buffer. The resulting cDNA fragments were purified using the QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), followed by end repair, poly(A) tailing, and ligation to Illumina sequencing adapters. The adapter-ligated cDNA libraries were size-selected by agarose gel electrophoresis, amplified by PCR, and subjected to high-throughput sequencing on the Illumina HiSeq2500 platform (Illumina, San Diego, CA, USA) at Gene Denovo Biotechnology Co. (Guangzhou, China). Sequencing data were processed to quantify gene abundance, followed by pathway enrichment analysis and Gene Ontology (GO) functional enrichment analysis of differentially expressed genes. These analyses were conducted using established bioinformatics pipelines and software tools.