The KAS-ATAC assay provides a method to capture genomic DNA fragments that are simultaneously physically accessible and contain single-stranded DNA (ssDNA) bubbles. These are characteristic features of two of the key processes involved in regulating and expressing genes-on one hand, the activity of -regulatory elements (cREs), which are typically devoid of nucleosomes when active and occupied by transcription factors, and on the other, the association of RNA polymerases with DNA, which results in the presence of ssDNA structures. Here, we present a detailed protocol for carrying out KAS-ATAC as well as basic processing of KAS-ATAC datasets and discuss the key considerations for its successful application. Key features • Allows mapping of simultaneously accessible and ssDNA-containing DNA fragments. • Describes the execution of N3-kethoxal labeling and transposition of native chromatin. • Describes the pulldown of biotin-labeled DNA fragments and library generation. • Describes basic KAS-ATAC data processing steps.