The study introduces Omni-ATAC, an improved ATAC-seq protocol that enhances chromatin accessibility profiling by reducing background noise and improving data quality. This protocol is applicable across various cell types, tissues, and archival frozen samples. It enables the analysis of chromatin accessibility in distinct human brain regions and facilitates the study of personal regulomes in tissue context. The protocol includes modifications such as the use of multiple detergents to improve permeabilization and remove mitochondria, a post-lysis wash step to increase library complexity, and the use of phosphate-buffered saline to enhance the signal-to-background ratio. The Omni-ATAC protocol generates data with higher quality and consistency compared to standard ATAC-seq, Fast-ATAC, and DNase-seq methods, as demonstrated by higher peak detection rates and improved signal-to-background ratios. It also allows for the analysis of chromatin accessibility in small cell numbers and frozen tissues, including snap-frozen pellets and primary human keratinocytes. The protocol was tested on various tissues, including frozen brain samples and mouse tissues, and showed improved data quality. The study also demonstrated the application of Omni-ATAC to clinically relevant frozen tissues, such as brain and cancer tissues, and enabled the generation of chromatin accessibility profiles from frozen tissue sections. The protocol was combined with histopathology to enable the analysis of chromatin accessibility in relation to histological features, such as glial and neuronal cell abundance. The results showed that the Omni-ATAC protocol provides a robust and broadly applicable platform for generating high-quality chromatin accessibility data, which can be used for clinical diagnosis and research. The study highlights the potential of epigenomic information in clinical applications and demonstrates the utility of the Omni-ATAC protocol in improving the accuracy and reliability of chromatin accessibility profiling.The study introduces Omni-ATAC, an improved ATAC-seq protocol that enhances chromatin accessibility profiling by reducing background noise and improving data quality. This protocol is applicable across various cell types, tissues, and archival frozen samples. It enables the analysis of chromatin accessibility in distinct human brain regions and facilitates the study of personal regulomes in tissue context. The protocol includes modifications such as the use of multiple detergents to improve permeabilization and remove mitochondria, a post-lysis wash step to increase library complexity, and the use of phosphate-buffered saline to enhance the signal-to-background ratio. The Omni-ATAC protocol generates data with higher quality and consistency compared to standard ATAC-seq, Fast-ATAC, and DNase-seq methods, as demonstrated by higher peak detection rates and improved signal-to-background ratios. It also allows for the analysis of chromatin accessibility in small cell numbers and frozen tissues, including snap-frozen pellets and primary human keratinocytes. The protocol was tested on various tissues, including frozen brain samples and mouse tissues, and showed improved data quality. The study also demonstrated the application of Omni-ATAC to clinically relevant frozen tissues, such as brain and cancer tissues, and enabled the generation of chromatin accessibility profiles from frozen tissue sections. The protocol was combined with histopathology to enable the analysis of chromatin accessibility in relation to histological features, such as glial and neuronal cell abundance. The results showed that the Omni-ATAC protocol provides a robust and broadly applicable platform for generating high-quality chromatin accessibility data, which can be used for clinical diagnosis and research. The study highlights the potential of epigenomic information in clinical applications and demonstrates the utility of the Omni-ATAC protocol in improving the accuracy and reliability of chromatin accessibility profiling.