Iso seq of vg
Firstly, the first strand cDNA was synthesized from the polyA+ tail by NEBNexts reverse transcriptase. Then the enzymes terminal transferase activity added a few extra nucleotides to the 3 end of the cDNA. Subsequently template switching, extension and amplification were performed to generate double-stranded cDNA. The cDNA was ligated with universal hairpin adapters. After purification with magnetic beads , sequencing primer was annealed to the SMRTbell templates, followed by binding of the sequence polymerase to the annealed templates.