GSM9712643: F20_5; Mus musculus; RNA-Seq
Total RNA was extracted from forelimb joint tissues of DBA/1 male mice. After euthanasia by spinal cord dislocation, both forelimbs were dissected by transecting at the distal humerus to retain the entire forearm, wrist, and paw. Skin and excess muscle were carefully removed, preserving joint capsules, synovium, and periarticular soft tissues. Samples were rinsed in PBS, snap-frozen in liquid nitrogen, and stored at low temperature until processing. Total RNA was isolated under RNase-free conditions. RNA concentration was measured using a Qubit 4.0 fluorometer or microplate reader, and RNA integrity was assessed with a Qsep400 bioanalyzer. Only RNA samples that passed quality control were used for subsequent library preparation and sequencing. mRNA-seq libraries were prepared from high-quality total RNA under RNase-free conditions. Polyadenylated mRNA was first enriched from total RNA, followed by fragmentation and first- and second-strand cDNA synthesis. The resulting double-stranded cDNA underwent end repair, A-tailing, and ligation of sequencing adapters. Adapter-ligated fragments were purified and amplified by PCR to generate the final libraries. Library concentration was measured with Qubit, and insert size distribution was assessed to evaluate library quality. Qualified libraries were converted to DNA nanoballs (DNBs), loaded onto the sequencing flow chip, and subjected to paired-end high-throughput sequencing on an MGI platform.