CapZyme-Seq: A 5′-RNA-Seq Method for Differential Detection and Quantitation of NAD-Capped and Uncapped 5′-Triphosphate RNA

Nucleoside-containing metabolites such as the oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD(+) and NADH), 3′-desphospho-coenzyme A (dpCoA), and flavin adenine dinucleotide (FAD) can be incorporated as RNA 5′ end caps by serving as non-canonical initiating nucleotides (NCINs) for transcription initiation by RNA polymerase. We recently reported “CapZyme-seq,” a 5′-RNA-seq method that enables the differential detection and quantitation of relative yields of NCIN-capped RNA and uncapped 5′-triphosphate RNA. Here we provide the protocol for constructing cDNA libraries for CapZyme-seq. For complete information on the generation and use of this protocol, please refer to Vvedenskaya et al. (2018a).