Lisa N Hansen, Mary T Couvillion, Erik McShane, Nadezhda Azbukina, Barbara Treutlein, L Stirling Churchman
Steady-state RNA abundance measurements mask the synthesis and decay rates that shape gene expression. Analog intrinsic recoding sequencing (AIR-seq) repurposes the base-pairing properties of N4-hydroxycytidine (NHC) to mark newly synthesized RNA with C-to-T and T-to-C mismatches in standard RNA-seq libraries, eliminating chemical conversion and enrichment common to RNA metabolic labeling methods. NHC-AIR-seq resolves bulk and single-cell RNA dynamics, improves RNA velocity inference, reveals hidden regulation, and brings RNA kinetics into routine transcriptomic workflows.