ONE-seq: epitranscriptome and gene-specific profiling of NAD-capped RNA

The hub metabolite, nicotinamide adenine dinucleotide (NAD), can be used as an initiating nucleotide in RNA synthesis to result in NAD-capped RNAs (NAD-RNA). Since NAD has been heightened as one of the most essential modulators in aging and various age-related diseases, its attachment to RNA might indicate a yet-to-be discovered mechanism that impacts adult life-course. However, the unknown identity of NAD-linked RNAs in adult and aging tissues has hindered functional studies. Here, we introduce ONE-seq method to identify the RNA transcripts that contain NAD cap. ONE-seq has been optimized to use only one-step chemo-enzymatic biotinylation, followed by streptavidin capture and the nudix phosphohydrolase NudC-catalyzed elution, to specifically recover NAD-capped RNAs for epitranscriptome and gene-specific analyses. Our data describes more than a thousand of previously unknown NAD-RNAs in the mouse liver and reveals epitranscriptome-wide dynamics of NAD-RNAs with age.ONE-seq empowers the identification of NAD-capped RNAs that are responsive to distinct physiological states, facilitating functional investigation into this modification. Overall design: ONE-seq of total RNAs (18-mon-old mouse livers) with spike-in RNA that had different ratio of NAD-RNA (0, 1, 5, 10%); ONE-seq with or without NudC treatment of mouse livers (12-mon-old); NAD-RNA profiles of young (2-mon-old) and aged (18-mon-old) mouse livers were generated by deep sequencing, in quadruplicate, using Illumina Novaseq.