Cells with variable mRNA content (blue lines) shown for an example gene. Cells are incubated in the presence of the uracil analogue 4sU for a set amount of time (4h). Transcripts that are produced during that period (red) become labelled with 4sU, which is incorporated instead of uracil. During the incubation period, natural mRNA decay also takes place (dashed lines). Following cell barcoding and RNA extraction, the RNA is chemically treated, resulting in the modification (alkylation) of the 4sU moieties incorporated into all labelled transcripts (U S ). In turn, this introduces T-to-C base flip mutations at the points of 4sU incorporation during the first stage of cDNA library preparation (reverse transcription), which are subsequently detected by sequencing.
