Supplementary Figure 5: Cycle dependency of DSB clustering and repair in DIvA cells. | Nature Structural & Molecular Biology

Supplementary Figure 5: Cycle dependency of DSB clustering and repair in DIvA cells.

From: Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes

Supplementary Figure 5

a. For cell cycle analysis in high throughput microscopy analyses, G1 and G2 cells are sorted based on Hoechst intensity. An example of Hoechst distribution for an experiment is shown. b. Averaged foci number (left panel) and foci size (middle panel) in G1 and G2 nuclei using 4 independent experiments (>1000 nuclei in each replicate). Foci number and foci size were set to 1 in G1. Mean and s.e.m are shown for n=4, independent experiments. * p<0.05; **** p<0.001 (one sample t-test). Right panel shows the average ratio between foci size and foci number per cells, hereafter named as the « clustering index » (n=4, independent experiments). c. Experimental pipeline used to analyze repair kinetics of clustered and unclustered DSBs in G1 and cycling cells (Fig. 5a): DIvA cells were first either arrested in G1 using lovastatin treatment for 48h or left untreated (cycling). Cells were next treated 4hours with 4OHT to induce DSBs, and further treated with auxin (IAA) to induce enzyme degradation and repair. Cells were collected at 0h, 2h, 8h, and 14h after IAA addition, and subjected to FACS analysis (Fig. S5d) and cleavage assay (Fig. 5a). Briefly, DNA was extracted and ligated to a biotinylated double strand oligonucleotide cohesive with AsiSI sites. After strepatividin purification, pulled down DNA is measured by qPCR at selected DSBs. Percent of purified DNA compared to input reflects the extent of cleavage of a given DSB in the cell population at a given time point. d. FACS profiles indicating the cell cycle distribution at each time point collected for repair kinetics analysis. e. Western blot was performed in cycling and G1-arrested cells (following lovastatin treatment) before and after damage induction and 2h after IAA addition in order to verify that enzyme degradation following IAA addition is as efficient in both conditions.

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