Enzymatic modification of the initiating nucleotide incorporated by the RNA polymerase identifies genome-wide transcription start sites in E. coli.. Capable-seq : A novel strategy for investigating transcriptomes by capturing primary RNA transcripts.

The initiating nucleotide found at the 5’ end of primary transcripts has a distinctive triphosphorylated end that distinguishes these transcripts from all other RNA species. Recognizing this distinction is key to deconvoluting the primary transcriptome from the plethora of processed transcripts that confound analysis of the transcriptome. We have developed Cappable-seq that specifically captures primary RNA transcripts by enzymatically modifying the 5’ triphosphorylated end of RNA with a selectable tag. In this study, we applied Cappable-seq to E. coli for the identification of transcription start sites (TSS) genome-wide at single base resolution with unprecedented level of specificity. Such specificity achieves up to 50 fold enrichment of primary transcripts allowing the identification of 16359 TSS of which 41% are novel. Beyond TSS determination, Cappable-seq depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per transcript enabling digital profiling of gene expression.