SRP118708 PRJNA411835 Detection and quantification of NAD-capped RNAs generated in vitro and in vivo Nucleoside-containing metabolites such as NAD+ can be incorporated as “5'' caps” on RNA by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase (RNAP). Here, we report “CapZyme-Seq,” a high-throughput-sequencing method that employs NCIN-decapping enzymes NudC and Rai1 to detect and quantify NCIN-capped RNA. By combining CapZyme-Seq with multiplexed transcriptomics, we determine efficiencies of NAD capping by Escherichia coli RNAP for ~16,000 promoter sequences. The results define preferred transcription start-site (TSS) positions for NAD capping and define a consensus promoter sequence for NAD capping: HRRASWW (TSS underlined). By applying CapZyme-Seq to E. coli total cellular RNA we establish that sequence determinants for NCIN capping in vivo match the NAD -capping consensus defined in vitro, and we identify and quantify NCIN-capped small RNAs. Our findings define the promoter-sequence determinants for NCIN capping with NAD and provide a general method for analysis of NCIN capping in vitro and in vivo.