Assay for Transposase-Accessible Chromatin Sequencing (ATAC-seq) of Cancer Cells in Culture.

The Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) is a powerful and widely-used technique for chromatin accessibility profiling due to its low input requirement, simple protocol, high sensitivity, and ability to study single cells. ATAC-seq is a powerful tool in cancer epigenetics research that has enabled the scrutiny of gene regulatory mechanisms, aided the identification of therapeutic targets, and in the exploration of anticancer immunity, thereby advancing our understanding of cancer biology. The protocol includes five main steps: cell isolation and nuclei preparation, Tn5 tagmentation, PCR amplification, library sequencing, and data analysis. Here, we have outlined a comprehensive ATAC-seq protocol that is utilized by our laboratories, along with detailed troubleshooting steps at each point, in the hopes that it will aid other researchers in utilizing this tool to the fullest capability. We also describe bioinformatics pipelines for processing the raw sequence data and identifying regulatory regions by differential accessibility analysis, as well as more advanced methods for the visualization of transcription factor binding sites.