This unit describes ATAC-seq, a method for mapping chromatin accessibility genome-wide using hyperactive Tn5 transposase. The method involves inserting sequencing adapters into accessible regions of chromatin, allowing for the inference of increased accessibility and transcription factor binding. ATAC-seq is a fast and sensitive alternative to DNase-Seq and MNase for assessing chromatin structure and composition. The protocol includes cell lysis, transposition, and amplification steps, with critical parameters such as cell number and collection methods affecting the quality of the results. ATAC-seq can be adapted for various cell types and species, and the method is optimized for human lymphoblastoid cells. The article also provides guidelines for library quality control and troubleshooting common issues.This unit describes ATAC-seq, a method for mapping chromatin accessibility genome-wide using hyperactive Tn5 transposase. The method involves inserting sequencing adapters into accessible regions of chromatin, allowing for the inference of increased accessibility and transcription factor binding. ATAC-seq is a fast and sensitive alternative to DNase-Seq and MNase for assessing chromatin structure and composition. The protocol includes cell lysis, transposition, and amplification steps, with critical parameters such as cell number and collection methods affecting the quality of the results. ATAC-seq can be adapted for various cell types and species, and the method is optimized for human lymphoblastoid cells. The article also provides guidelines for library quality control and troubleshooting common issues.