GSM9796276: Subchondral bone from DMM-operated wild-type mouse (8 weeks post-surgery), biological replicate 4; Mus musculus; RNA-Seq
Total RNA was extracted from frozen tibial subchondral bone tissues using TRIzol reagent (Invitrogen, #15596026) combined with the RNeasy Mini Kit (Qiagen, #74104) following the manufacturer's instructions. Briefly, bone tissues were ground into fine powder in liquid nitrogen, lysed in TRIzol, and centrifuged to remove debris. The aqueous phase was collected, mixed with 70% ethanol, and loaded onto RNeasy spin columns. On-column DNase I digestion (Qiagen, #79254) was performed to eliminate genomic DNA contamination. RNA concentration and purity were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher), and RNA integrity was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies). Only samples with RNA Integrity Number (RIN) ≥ 8.0 were used for library construction. mRNA-seq libraries were constructed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB, #E7770) according to the manufacturer's protocol. Briefly, 1 μg of total RNA per sample was used for poly(A) mRNA enrichment using oligo(dT) magnetic beads. The enriched mRNA was fragmented into 200-300 bp fragments at 94°C for 15 minutes. First-strand cDNA was synthesized using random hexamer primers and reverse transcriptase, followed by second-strand cDNA synthesis with dUTP instead of dTTP. The double-stranded cDNA was subjected to end repair, dA-tailing, and ligation of NEBNext adaptors with unique barcodes. After size selection (250-350 bp) using AMPure XP beads (Beckman Coulter), the libraries were amplified by PCR for 12-15 cycles. The final libraries were quantified using a Qubit 3.0 fluorometer (Thermo Fisher) and validated for size distribution using an Agilent 2100 Bioanalyzer.