RNA extraction--RNA quality inspection-- Reverse transcription into cDNA-- Equimolar mixing of cDNA purified with two magnetic beads-- library preparation--DNA damage repair and End repair-Add adapter--Enzyme digestion removes libraries with add adapter failed--Purified library with magnetic beads--library quality inspection-Sequencing--Use isoseq3 to process Subreads to generate high quality isoform--Bioinformatics analysis