RNA-Seq of TSS-barcode amplicons from in vitro Tx reactions
Starting with RNA harvested from the in vitro transcription reactions, the locations of transcriptional start sites (TSSs) were determined by treating the RNA with TURBO DNase to remove template DNA, purifying, treating with RNA 5 polyphosphatase (Epicentre) to remove 5 phosphates, purifying, ligating the RNA to an 5 RNA adapter using T4 RNA Ligase (NEB), and purifying again. The treated RNA product was then converted to cDNA by treating with SuperScript IV (Invitrogen) and the transcript-specific primer 2. PCR was then carried out using 10ng of cDNA as template, primers 2 and 5, and 30 cycles of amplification. The linear DNA product was then circularized by first treating it with T4 Polynucleotide Kinase, followed by ligating using T4 DNA Ligase. Monomer DNA circles were gel extracted. PCR was then carried out to generate the barcode-TSS amplicon library, using primers 3 and 6 in 25 cycles of amplification. The barcode-TSS amplicon library was then sequenced using Illumina HiSeq 2500 (150 bp, paired-end).