RNA-Seq of mus musculus
Magnetic beads with oligo (dT) were utilized for mRNA enrichment. The isolated mRNA is fragmented using a breaking buffer, and reverse transcription is carried out to synthesize double-stranded cDNA. The synthesized double-stranded DNA is then end-repaired and phosphorylated to create a 3-overhanging "A" sticky end, which is li-gated with a bubble-shaped adaptor with a 3-overhang "T". Subsequently, using specific primers, the ligation products are amplified via PCR, followed by purification using Ampure XP Beads and resuspension in EB buffer. Quality assessment of the PCR products was conducted using the Agilent 2100 Bioanalyzer. The PCR products are first denatured to single-stranded DNA, then circularized into a single-stranded circular DNA library using a bridging primer. This single-stranded circular DNA was prepared as the defini-tive library. The library was then amplified using phi29 to create DNA nanoballs (DNBs), each containing over 300 molecules. These DNBs were loaded into a nanoarray and paired-end 150 base reads were generated on the MGIseq2000 platform (BGI, Shenzhen, China).