Fig. 1: System to distinguish gradual and acute mutational processes in mammalian cells.

a, Model system to interrogate gradual (ROS, blue) and acute mutation pressure (UV exposure, brown) on the mammalian genome. Triangles represent gradual accumulation of ROS mutations over several cell generations, whereas boxes represent fixed mutations in the genome after a specific cell division event. Schematic below depicts the experimental method from standard cell culture, exposure to UV, penning single cells, splitting two sister cells into separate pens after the first mitosis and finally proliferation/export. b, Images of single penned cells and FUCCI fluorophores imaged on the Phenomex Lightning platform (left), splitting mitotic sisters with light cages (middle), as well as the expanded populations (right). WGS, whole-genome sequencing. c, A representative panel of cells (n = 90) imaged to ascertain the intensity of red and green fluorophores in 3-h intervals after penning. Cell cycle designation to the left of each cluster was determined by the fluorophore intensity at the time of penning seen in column 1 and denoted by the gray border. Subsequent heatmap columns represent 3 hour imaging timepoints of the same cell. Cell cycle color key circle is below. Models to the right implicate theoretical strand-specific (blue/yellow) distribution of DNA damage (red triangles) depending on the cell cycle phase at the time of UV treatment (timepoint 0).