SPAAC-NAD-seq, a sensitive and accurate method to profile NAD + -capped transcripts

Significance The m 7 G cap is the canonical RNA cap in eukaryotes, but other noncanonical RNA caps exist, including the NAD + cap. NAD captureSeq has been widely used to profile NAD + -capped RNAs (NAD-RNAs) in prokaryotes and eukaryotes. However, NAD captureSeq reacts at a low level with m 7 G-RNAs and introduces copper ions that cause RNA fragmentation, resulting in reduced sensitivity and loss of full-length sequence information. To address these issues, we developed the copper-free SPAAC-NAD-seq, which utilizes the strain-promoted azide–alkyne cycloaddition reaction to capture NAD-RNAs followed by high-throughput sequencing. Compared to NAD captureSeq, SPAAC-NAD-seq is more sensitive and retains full-length sequence information. This optimized technique provides a useful tool to profile NAD-RNAs in prokaryotes and, when combined with m 7 G-RNA depletion, in eukaryotes.