RNA-seq of C2-12-17
The RNA input used for MeRIP-seq was subjected to mRNA-seq analysis. mRNA was isolated using EpiTM mRNA Capture Beads. The captured mRNA was then used for library construction with the EpiTM mRNA Library Fast kit, according to the manufacturers instructions. First, the mRNA was fragmented with the addition of 3 Frag/Prime Buffer, followed by incubation at 94C for 6 min and immediate cooling on ice. For first-strand cDNA synthesis, Actinomycin D (5 mg/mL), 1st Strand Buffer 2, RT Enzyme, and RI were added, and the reaction was sequentially carried out at 25C for 10 min, 42C for 15 min, and 70C for 15 min. The reaction was then placed on ice. Second-strand synthesis was performed by the addition of 2nd Strand Buffer 2 (with dUTP) and 2nd Strand Enzyme Mix, followed by incubation at 16 for 30 min and 72 for 15 min. Subsequently, adapter ligation was conducted by the addition of Ligation Buffer 3, EPI DNA Ligase, and Adapter, followed by incubation at 22 for 15 min. The ligation products were purified using Epi DNA Clean Beads. Library fragments were enriched by PCR amplification using 2HiFi PCR Mix, Heat-labile UDG, and Index primers. The final libraries were purified and recovered using Epi DNA Clean Beads. Library quality and fragment size distribution were analyzed using a Bioptic Qsep100 Analyzer to ensure they matched the expected size range. Sequencing was performed on the NovaSeq high-throughput sequencer using the PE150 sequencing mode.