GSM9398961: C2C12 cells,control,rep1,control; Mus musculus; RNA-Seq
Total RNA was extracted from C2C12 cells using TRIzol reagent (Invitrogen/Servicebio) according to the manufacturer's instructions. Cells were lysed directly in the culture dish by adding 1 mL of TRIzol and homogenized by pipetting. After addition of chloroform, the mixture was centrifuged to separate the aqueous phase containing RNA. RNA was then precipitated with isopropanol, washed with 75% ethanol, and finally dissolved in RNase-free water. RNA concentration and purity (A260/280 and A260/230 ratios) were measured using a NanoDrop spectrophotometer. RNA integrity was further assessed by agarose gel electrophoresis or an Agilent 2100 Bioanalyzer to ensure suitability for downstream sequencing. Sequencing libraries were prepared using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® following the manufacturer's instructions for non-strand-specific library preparation. Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was performed using divalent cations under elevated temperature. First-strand cDNA was synthesized using random hexamer primers and M-MuLV Reverse Transcriptase (RNase H-). Second-strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. The double-stranded cDNA fragments were then subjected to end-repair, A-tailing, and ligation with NEBNext Adaptors. To select cDNA fragments of approximately 250~300 bp, the library fragments were purified with the AMPure XP system (Beckman Coulter). PCR amplification was performed using Phusion High-Fidelity DNA polymerase and universal/index primers. Finally, PCR products were purified, and library quality was assessed on the Agilent Bioanalyzer 2100 system.