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).