The authors present an improved ATAC-seq protocol called Omni-ATAC, which enhances signal-to-background ratio and information content. This protocol is designed to work across multiple applications, including archival frozen tissue samples and 50-μm sections, and is suitable for diverse cell lines, tissue types, and archival frozen samples. The Omni-ATAC protocol improves data quality by reducing the number of sequencing reads mapping to mitochondrial DNA, increasing the percentage of reads mapping to peaks of chromatin accessibility, and generating more unique fragments per input cell. It also enables the interrogation of personal regulomes in tissue context and translational studies. The protocol is demonstrated to be effective in various cell types and contexts, including snap-frozen pellets, primary human keratinocytes, and clinically relevant frozen tissues such as brain and cancer samples. The authors show that Omni-ATAC can generate high-quality chromatin accessibility profiles from frozen tissues, revealing disease-associated DNA element activities in distinct human brain structures. The protocol also facilitates the interpretation of genome-wide association study (GWAS) results by mapping putative brain-disease-relevant single-nucleotide polymorphisms to noncoding regions. Overall, the Omni-ATAC protocol provides a robust and broadly applicable platform for generating high-quality chromatin-accessibility data, making it more universally comparable and facilitating the use of ATAC-seq in challenging cell lines, rare primary cells, and clinically relevant frozen tissues.The authors present an improved ATAC-seq protocol called Omni-ATAC, which enhances signal-to-background ratio and information content. This protocol is designed to work across multiple applications, including archival frozen tissue samples and 50-μm sections, and is suitable for diverse cell lines, tissue types, and archival frozen samples. The Omni-ATAC protocol improves data quality by reducing the number of sequencing reads mapping to mitochondrial DNA, increasing the percentage of reads mapping to peaks of chromatin accessibility, and generating more unique fragments per input cell. It also enables the interrogation of personal regulomes in tissue context and translational studies. The protocol is demonstrated to be effective in various cell types and contexts, including snap-frozen pellets, primary human keratinocytes, and clinically relevant frozen tissues such as brain and cancer samples. The authors show that Omni-ATAC can generate high-quality chromatin accessibility profiles from frozen tissues, revealing disease-associated DNA element activities in distinct human brain structures. The protocol also facilitates the interpretation of genome-wide association study (GWAS) results by mapping putative brain-disease-relevant single-nucleotide polymorphisms to noncoding regions. Overall, the Omni-ATAC protocol provides a robust and broadly applicable platform for generating high-quality chromatin-accessibility data, making it more universally comparable and facilitating the use of ATAC-seq in challenging cell lines, rare primary cells, and clinically relevant frozen tissues.