TimeLapse-seq: adding a temporal dimension to RNA sequencing through nucleoside recoding

RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report TimeLapse-seq, which uses oxidative-nucleophilic-aromatic substitution to convert 4-thiouridine into cytidine analogs, yielding apparent U-to-C mutations that mark new transcripts upon sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications and reveals RNA dynamics and induced differential expression concealed in traditional RNA-seq. RNA isolated from cells +/- s4U feed was subjected to oxidative-nucleophilic-aromatic-substitution chemistry and sequenced. All sequencing was performed in duplicate per condition assessed.