GSM9473001: db/m1; Mus musculus; RNA-Seq
Total RNA was isolated from mouse tissue samples using a commercially available RNA extraction kit (e.g., Trizol reagent or column-based RNA purification kit) following the manufacturer's standard protocols. Briefly, the collected samples were homogenized in lysis buffer to fully release intracellular RNA, followed by phase separation with chloroform to remove protein and genomic DNA contaminants. The aqueous phase containing RNA was then precipitated with isopropanol, and the RNA pellet was washed with 75% ethanol, air-dried, and resuspended in RNase-free water. The quality and integrity of the extracted total RNA were evaluated using a NanoDrop spectrophotometer (to assess the A260/A280 ratio, with an optimal range of 1.8–2.1) and an Agilent 2100 Bioanalyzer (to determine the RNA Integrity Number, RIN ≥ 7.0). Only high-quality RNA samples that passed the above quality control criteria were subjected to subsequent mRNA enrichment and library construction. A strand-specific mRNA-seq library was constructed using the enriched mRNA from total RNA samples. First, eukaryotic mRNA was purified and enriched from total RNA using magnetic beads conjugated with Oligo(dT) to specifically bind the poly(A) tails of mRNA molecules. The purified mRNA was fragmented into short fragments (approximately 200–300 bp) by adding fragmentation buffer under thermostatic conditions. Using these fragmented mRNAs as templates, the first strand of cDNA was synthesized with random hexamer primers, followed by the synthesis of the second strand of cDNA using DNA polymerase I, RNase H, dNTPs, and buffer. The double-stranded cDNA products were then subjected to end repair, 3' end adenylation, and ligation of sequencing adapters. After adapter ligation, the cDNA fragments of target size were selected by magnetic bead purification, and the resulting cDNA library was amplified by PCR with limited cycles to enhance library yield. Finally, the constructed library was quantified using a Qubit fluorometer and its insert size was verified using an Agilent 2100 Bioanalyzer. Qualified libraries with consistent insert size and sufficient concentration were used for high-throughput sequencing.
GEO
GSM9473001_r1