Fig. 1
Global sequencing of newly synthesised RNA in single cells. a Illustration of the NASC-seq methodology. In brief, alkylation is performed on RNAs immobilised on beads for the subsequent wash before proceeding with standard PCR, tagmentation, and construction of sequencing libraries. Modified 4sU is marked in ellipses, nucleotide sequences corresponding to oligo-dT primers are shown in light grey, TSO, and ISPCR primers in dark grey, sequences added during tagmentation are indicated by blue bars. b Observed conversion rates in K562 cells labelled with 4sU (50μM, 3 h; red, 16 cells) or unlabelled (grey, 14 cells) on the positive strand within genes. T–C (tC) conversions are significantly (P-value = 1.375e−08, Mann–Whitney U-test, two-sided) increased in cells labelled with 4sU. Lowercase and uppercase letters indicate original and new base identities, respectively. The line in the boxplot indicates the median value, the two hinges display the first and third quartiles. The whiskers range from the hinges to the highest or lowest point that is no further than 1.5 times the interquartile range. c Scatter plots showing the total number of sequenced reads (x-axis) against the number of reads with T–C conversions (y-axis) for the MYC, PDLIM5, and GAPDH genes in 4sU labelled (50 μM, 1 h) and unlabelled cells. d Signal to noise estimated as pc divided by pe for K562 cells exposed to 50 μM for 1 h (n = 106) compared to K562 cells that were not exposed to 4sU (n = 44). Median, hinges, and whiskers are shown as in b. e Scatter plots showing newly synthesised (new) RNA inferred using the mixture-model (blue) or based on conversions directly (red) against total RNA (i.e. number of reads) for the same genes and 4sU-labelled cells from c. Source data for panels b and d are provided as a Source Data file
