Random transposon insertion mutagenesis system identifies potential transgene locations in CVA21 genome

Here we performed random transposon insertion mutagenesis to randomly insert a 15 base pairs (bp) sequence (TGCGGCCGCANNNNN, where NNNNN represents the duplicated target site) to the CVA21 cDNA to create a plasmid library, which was then transcribed in vitro to make an RNA library. SK-Mel-28 cells were transfected with the RNA library to generate a recombinant virus library. vRNA purified from the virus library was sequenced by standard RNA-seq deep sequencing, to identify tolerable insert locations on CVA21 genome carrying the 15 nt insertion.