RNA-Seq of barcoded amplicons from in vitro Tx reactions, repl 1
For quantification of RNA variant levels after transcription (RNA-Seq), cDNA first-strand synthesis reactions were first carried out on the harvested RNA, using 200 units of SuperScript IV (Invitrogen), 1X buffer, and transcript-specific primer 2. PCR was carried out to generate barcode-containing amplicons (121 bp), using 5 ng of cDNA as template, primers 2 and 3, and 25 cycles of amplification, followed by next-generation sequencing (Illumina HiSeq 2500, 150 bp paired-end), barcode mapping, and barcode counting.