Extended Data Fig. 10: Hypomethylated CpG state is essential to retention element function.

(a) 5mC methylation status of individual CpG sites and their density within and surrounding retention elements on the EGFR ecDNA in GBM39 cells as measured in single-molecule long-read nanopore sequencing. (b) Viability of cells expressing CRISPRoff and a targeting guide cargo or non-targeting control over time. Cells were sorted at day 2 post-transfection and tracked until day 12, when no live targeted cells remained. Each line represents an independent biological replicate. (c) Counts of cells expressing CRISPRoff and a targeting guide cargo or non-targeting control guide RNA over time. Cells were sorted at day 2 post-transfection and tracked until day 12, when no live targeted cells remained. Each line represents an independent biological replicate. (d) Abundance of ecDNA following CpG methylation of retention elements by CRISPRoff at 5 days post-transfection compared to cells expressing a non-targeting control guide RNA in WGS coverage. (e) Representative image showing ecDNA foci lost from the nucleus in an interphase GBM39 cell 5 days after transfection with CRISPRoff and a guide cargo targeting retention elements (n = 50 image positions). Scale bar, 10 µm. (f) Abundance of nuclear ecDNA measured by nuclear EGFR DNA FISH signal at 5 days after transfection of CRISPRoff and guide cargo targeting retention elements compared to cells expressing a non-targeting control guide RNA. P-value computed using two-sided two-sample Kolmogorov-Smirnov test. (g) Mean cell trajectories of methylated retention element plasmid (n = 51 cells) or ecMYC DNA signal colocalization with chromosomes throughout mitosis. Mean cell trajectories include all time points with more than 3 cells. Measurements for the control and unmethylated retention element plasmid conditions are reproduced from Fig. 3d. Error bars show s.e.m. P-values determined by two-sided paired t-test of the means.