Epitranscriptome analysis of NAD-capped RNA by spike-in-based normalization and prediction of chronological age

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). We investigated the dynamics of NAD-modified epitranscriptome during human normal aging. A total of two independent cohorts were recruited: the discovery cohort (17 females and 18 males) aged 23-67 years and the validation cohort (14 females and 12 males) aged 23-67 years. Peripheral blood mononuclear cells (PBMCs) were isolated from blood drawn from participants, and used for NAD-RNA profilings. Combined, we subjected 10 μg total RNAs from human PBMCs mixed with 40 μg total RNAs from Drosophila, 0.1 ng spike-in 2 RNAs, and 0.1 ng spike-in 3 RNAs to NAD-RNA sequencing, followed by enONE computational analysis of NAD-RNA profiles.