ATAC-seq is a method for genome-wide mapping of chromatin accessibility. It uses hyperactive Tn5 transposase to insert sequencing adapters into accessible chromatin regions, allowing for the identification of accessible regions, transcription factor binding sites, and nucleosome positions. It is a fast and sensitive alternative to DNase-seq and MNase for chromatin accessibility and nucleosome mapping. The method is compatible with various cell separation techniques and works across many cell types and species. The protocol involves cell lysis, transposition, and amplification. Key parameters include cell number, which affects fragment size and library complexity. PCR amplification is optimized to reduce bias and ensure library quality. Libraries are quantified using qPCR and sequenced on Illumina platforms. ATAC-seq provides high-resolution data on chromatin accessibility and is useful for studying regulatory elements. The method is efficient, with a total time of about 3 hours, and can generate up to 50–100 million unique fragments per reaction. Troubleshooting involves optimizing cell number and lysis methods. The results show that approximately half of the reads are sub-nucleosomal in length, with a high concentration of reads in accessible regions. ATAC-seq is a valuable tool for epigenetic studies and chromatin analysis.ATAC-seq is a method for genome-wide mapping of chromatin accessibility. It uses hyperactive Tn5 transposase to insert sequencing adapters into accessible chromatin regions, allowing for the identification of accessible regions, transcription factor binding sites, and nucleosome positions. It is a fast and sensitive alternative to DNase-seq and MNase for chromatin accessibility and nucleosome mapping. The method is compatible with various cell separation techniques and works across many cell types and species. The protocol involves cell lysis, transposition, and amplification. Key parameters include cell number, which affects fragment size and library complexity. PCR amplification is optimized to reduce bias and ensure library quality. Libraries are quantified using qPCR and sequenced on Illumina platforms. ATAC-seq provides high-resolution data on chromatin accessibility and is useful for studying regulatory elements. The method is efficient, with a total time of about 3 hours, and can generate up to 50–100 million unique fragments per reaction. Troubleshooting involves optimizing cell number and lysis methods. The results show that approximately half of the reads are sub-nucleosomal in length, with a high concentration of reads in accessible regions. ATAC-seq is a valuable tool for epigenetic studies and chromatin analysis.