GSM9461124: Heart, SIC+AAVGata4, rep3; Mus musculus; RNA-Seq
Total RNA from the samples was isolated and purified using TRIzol (Thermo Fisher, Cat. No. 15596018) following the manufacturer's protocol. Subsequently, the quantity and purity of the total RNA were assessed using a NanoDrop ND-1000 spectrophotometer (NanoDrop, Wilmington, DE, USA), and RNA integrity was examined with a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Only RNA samples meeting the following criteria were used for downstream experiments: concentration > 50 ng/μL, RNA Integrity Number (RIN) > 7.0, and total RNA yield > 1 μg. PolyA-tailed mRNA was captured via two-round purification using oligo(dT) magnetic beads. The captured mRNA was fragmented by magnesium ion fragmentation kit at 94℃ for 5–7 minutes, followed by cDNA synthesis with reverse transcriptase. Double-stranded DNA was synthesized using E. coli DNA polymerase I and RNase H with dUTP incorporation, and then subjected to end-repair, dA-tailing, adapter ligation and magnetic bead-based purification. After double-stranded DNA digestion with UDG enzyme, the product was amplified by PCR for 8 cycles, generating a strand-specific library with an insert size of 300 bp ± 50 bp.