To control for the effects of focal amplification and assess ecDNA-specific transcriptional changes, we focused on a pair of isogenic colorectal cancer cell lines derived from the same patient: COLO320DM (MYC amplification on ecDNA, also called double minute (DM)) and COLO320HSR (chromosomal MYC amplification on homogeneously staining region (HSR)), which are nearly matched for amplicon copy number as revealed by whole-genome sequencing (WGS) (Extended Data Fig. 1a,d)11. Here, MYC is linked to colorectal cancer.