The Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) is a rapid and sensitive method for exploring the cancer immune epigenome. ATAC-seq captures open chromatin sites based on transposing the sequencing adapter directly into native chromatin in vitro. Here we describe an optimized protocol for ATAC-seq with four main steps: nucleus extraction, transposition, library amplification, sequencing and data analysis. The ATAC protocol has the advantages of low sample requirements, high speed, and applicability to a wide range of cell and tissue types.