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Background  Although extrachromosomal DNA (ecDNA) has been intensively studied for several decades, the mechanisms underlying its tumorigenic effects have been revealed only recently. In most conventional sequencing studies, the high-throughput short-read sequencing largely ignores the epigenetic status of most ecDNA regions except for the junctional areas.    Methods  Here, we developed a method of sequencing enzyme-accessible chromatin in circular DNA (CCDA-seq) based on the use of methylase to label open chromatin without fragmentation and exonuclease to enrich ecDNA sequencing depth, followed by long-read nanopore sequencing.    Results  Using CCDA-seq, we observed significantly different patterns in nucleosome/regulator binding to ecDNA at a single-molecule resolution.    Conclusions  These results deepen the understanding of ecDNA regulatory mechanisms.    Supplementary Information  The online version contains supplementary material available at 10.1186/s13072-021-00416-5.

Sequencing of methylase-accessible regions in integral circular extrachromosomal DNA reveals differences in chromatin structure