DNA-Seq of barcoded amplicons from in vitro Tx reactions, repl 2
For quantification of DNA variant levels in the expression plasmid library (DNA-Seq), PCR was first carried out on 10 ng of the expression plasmid library to generate promoter-barcode amplicons (847 bp long), using primers 1 and 2 and 25 cycles of amplification. The promoter-barcode regions were then condensed into shorter amplicons (271 bp) by treating the PCR product with T4 Polynucleotide Kinase (NEB), carrying out DNA circularization by ligation using T4 DNA Ligase (NEB), purifying the monomer DNA circles via gel extraction, and then PCR amplifying using primers 3 and 4 with 25 cycles. The promoter-barcode amplicon library was sequenced using Illumina HiSeq 2500 (150 bp, paired-end), followed by barcode mapping, promoter identification, and counting.