RNA-Seq of Mus musculus: adult female heart
Total RNA extraction was performed using the Trizol method; purity was assessed using NanoDrop One, and the integrity of total RNA was evaluated using Agilent 2100 or FA 471. For eukaryotic total RNA, mRNA enrichment and purification were conducted using the VAHTS mRNA Capture Beads kit. RNA fragmentation was performed by ion shearing to produce inserts of 250-450 bp. Using the fragmented RNA as a template, the first strand of cDNA was synthesized. The second strand of cDNA was then synthesized using the first-strand cDNA as a template, followed by double-stranded cDNA end repair and dA-tailing. Adapters were ligated, and magnetic bead purification was performed, with size selection for fragments of 250-350 bp. PCR amplification was conducted with dual-indexed primers. The library was purified using 0.9X magnetic beads to obtain the final library. Library quality was assessed using Qubit 4.0, LabChip, or FA, and ABI QuantStudio 12K qPCR. Sequencing was performed using the second-generation sequencing technology on the Illumina NovaSeq 6000 platform with paired-end (PE) sequencing.