Figure 1: Quantitative detection of natural and etoposide-induced DSBs.

(a) Schematic of BLISS. The workflow starts by either fixing cells onto a microscope slide or in a multi-well plate, or by immobilizing already fixed tissue sections onto a slide. DSB ends are then in situ blunted and tagged with dsDNA adapters containing components described in the boxed legend and in Supplementary Data 1. Tagged DSB ends are linearly amplified using in vitro transcription and the resulting RNA is used for Illumina library preparation and sequencing. (b) BLISS reads aligned to an SpCas9 on-target cut site (arrowhead) in the EMX1 gene. Light blue, guide sequence. Orange, PAM sequence. Dark blue, reads mapped to the minus strand. Red, reads mapped to the plus strand. (c) Estimated number of DSBs per cell in three replicates sequenced at increasing sequencing depth. Dashed line, hyperbolic interpolation. (d) Number of DSB locations in etoposide-treated versus control U2OS cells by filtering on the minimum number of UMIs per DSB location. (e) Fraction of DSB locations mapped around the transcription start sites (TSS) in control versus etoposide-treated U2OS cells as a function of the minimum number of UMIs per DSB location. Dashed lines, linear interpolation. Colour shades, 95% confidence intervals. (f) For BLISS on mouse liver, mapping of sequenced DSB ends found in the top 10% (red) and bottom 10% (blue) of expressed genes in the mouse liver. n, number of biological replicates. Dots, mean value. Whiskers, min-max range. Dashed lines, spline interpolation. (g) Percentage of sequenced DSB ends mapped in a ±1 kb interval around the TSS for each inter-decile interval of gene expression in mouse liver. FPKM, fragments per kilobase of transcript per million mapped reads. n, number of biological replicates. Bars, mean value. Whiskers, min–max range. (h) Number of sequenced DSB ends mapped per kilobase inside the gene body of the top 10% and bottom 10% expressed genes in mouse liver. n, number of biological replicates. Whiskers, 2.5–97.5 percentile range. P, Mann–Whitney test.