RNA sequencing (RNA-seq) offers a snapshot of cellular RNA populations, but not temporal information about the sequenced RNA. Here we report a protocol for 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. The steps of the protocol are (1) treating samples with 4-thiouridine and isolating the total RNA, (2) a simple chemical treatment of the labeled RNA with an oxidant and amine under optimized conditions, and (3) isolation of the converted RNA which can then be subjected to sequencing. TimeLapse-seq is a single-molecule approach that is adaptable to many applications.