RNA-seq of mus musculus: adult mice spleen
Total RNA was extracted using Trizol reagent. High-quality RNA samples with RIN number > 7.0 were used to construct sequencing library. Following purification, the mRNA was fragmented into short fragments using divalent cations under elevated temperature. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA library by SuperScript II Reverse Transcriptase. An A-base was added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Dual-index adapters were ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, the ligated products were amplified with PCR.