Fig. 1: Pervasive transcription on ecDNA drives ssDNA accumulation.

a, Schematic of relevant genomic assays. b, Read density of genomic assays in COLO320DM and COLO320HSR in total counts per million (CPM) within the ecDNA intervals (amplicon boundaries defined in Extended Data Fig. 1). KAS-seq read density is shown as CPM of the KAS-seq relative to CPM of the input of total DNA after fragmentation but before biotin enrichment for ssDNA signals. The mean of two biological replicates is shown for GRO-seq, Ribo-Zero and KAS-seq; a single replicate is shown for WGS. c, Genome tracks highlighting two regions within the ecDNA interval. H3K36me3 chromatin immunoprecipitation followed by sequencing (ChIP–seq) is displayed as log₂ of input-normalized coverage. d, Metagene heatmap plot visualization of GRO-seq, Ribo-Zero RNA sequencing (RNA-seq) and log₂ of input-normalized coverage of KAS-seq within the ecDNA interval. All plots are anchored at the transcription start site (TSS) of combined transcribed regions as identified by HOMER using both biological replicates of GRO-seq in COLO320DM and COLO320HSR. e, Metagene plot showing GRO-seq and H3K36me3 ChIP–seq coverage within the ecDNA interval. All plots are anchored at the GRO-seq TSS as identified by HOMER using both biological replicates. H3K36me3 ChIP–seq coverage is displayed as log₂(H3K36me3/input). f, KAS-seq peaks from two biological replicates in the ecDNA interval annotated by transcription status according to GRO-seq data and annotation status according to Gencode v.43. One representative biological replicate for each condition is visualized for c, d and e. chr amp, chromosomal amplification; RNA Pol II, RNA polymerase II.