Fig. 3

a Schematic representation of the protocol: 4sUTP (4-thiouridine-triphosphate) injection into zebrafish one-cell stage embryos, dechorionation, dissociation into single cells at gastrulation stage, and MeOH (methanol) fixation (see “Methods” section). Incorporated 4sUTP is converted in a SN2 reaction with iodoacetamide into a cytosine analog. The single-cell solution is then loaded onto 10× Genomics Chromium, and chemical labels lead to T-to-C conversions during reverse transcription. b Nucleotide mutation frequencies of a scSLAM-seq library after injecting 4sUTP or Tris and quality filtering of the data. c Histogram of T-to-C mutations in 4sUTP- and Tris-injected embryos. d UMAP representation of cells based on labeled RNA (left side) and unlabeled RNA (right side). For the latter, we imposed cell identities as determined on the basis of labeled RNA. e Marker gene expression of labeled cells in different cell types (color code as in d). Cell number per cluster was downsampled to equal numbers. f Transcript labeling efficiency in single cells in percent, projected on the UMAP representation for labeled RNA.