a, Total number of genes detected after scSLAM-seq across all four experimental conditions (uninfected and CMV-infected cells; two biological replicates) versus the total read counts per single cell. The horizontal line indicates a threshold below which cells were excluded from the analysis. b, Partition of reads devoted for host (cellular), viral, spike-in control (External RNA Controls Consortium (ERCC)) and mitochondrial genes (Mitoch) across all individual cells. c, Rates of nucleotide substitutions demonstrate efficient conversion rates in 4sU-treated single cells (4sU) compared with 4sU-naive cells (no4sU). This was true for reads originating from both cDNA strands (sense and antisense) as well as overlapping parts of the paired-end sequencing (overlap). d, As in c, zoomed into the range (y axis) 0 to 0.004. e, Number of genes per cell for which the NTR could be quantified with high precision (90% credible interval (CI) < 0.2) compared with the detected genes and reliably detected genes (TPM >10). f, Correlation between expression levels of bulk RNA-seq with the pooled scRNA-seq data for total, new and old RNA. Genes are coloured according to RNA half-life. Pearson’s correlation coefficient (R) and the number of genes used (n) are indicated. g, Identification of highly variable genes (magenta) using ERCC spike-ins to model the technical noise applied to total RNA (1% false discovery rate). Squared coefficients of variation (CV2) are plotted against the average normalized read counts for all cells that pass the quality-control filters. The solid pink line fits the average values for ERCC spike‐ins (blue dots)25. The dashed line marks the expected position of genes with 50% biological coefficient of variation. h, PCA (the two first components are depicted) of highly variably genes including the viral transcripts (infected: n = 2 replicates, 49 cells; uninfected: n = 2 replicates, 45 cells). i, Correlation of the percentage of viral reads with the distance to uninfected cells in the first two principal components as measured by logistic regression for the PCA in f (n = 2 replicates, 49 cells). j, As in i, for the PCA in Fig. 2a (n = 2 replicates, 49 cells).