The study introduces single-cell ATAC-seq (scATAC-seq), a method for mapping accessible chromatin in individual cells, which reveals the landscape and principles of cellular DNA regulatory variation. ScATAC-seq is an improvement on the existing ATAC-seq method, achieving over 500-fold higher sensitivity. The authors developed a microfluidic platform to capture and assay single cells, generating DNA accessibility maps from hundreds of individual cells. These maps closely resemble those from tens of millions of cells, providing insights into cell-to-cell variation. Accessibility variance is associated with specific trans-factors and cis-elements, and the study identifies combinations of trans-factors that either induce or suppress cell-to-cell variability. Additionally, the authors find sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of the cell cycle or transcription factor signaling evoke stimulus-specific changes in accessibility variance, demonstrating that variability is not solely dependent on the cell cycle. The study also reveals that trans-factors promote cell-type-specific chromatin accessibility variation genome-wide and that variation in chromatin accessibility in cis is highly correlated with previously reported chromosome compartments, suggesting a connection to higher-order chromosome folding. Overall, the findings provide new hypotheses about the regulatory mechanisms underlying single-cell heterogeneity.The study introduces single-cell ATAC-seq (scATAC-seq), a method for mapping accessible chromatin in individual cells, which reveals the landscape and principles of cellular DNA regulatory variation. ScATAC-seq is an improvement on the existing ATAC-seq method, achieving over 500-fold higher sensitivity. The authors developed a microfluidic platform to capture and assay single cells, generating DNA accessibility maps from hundreds of individual cells. These maps closely resemble those from tens of millions of cells, providing insights into cell-to-cell variation. Accessibility variance is associated with specific trans-factors and cis-elements, and the study identifies combinations of trans-factors that either induce or suppress cell-to-cell variability. Additionally, the authors find sets of trans-factors associated with cell-type-specific accessibility variance across eight cell types. Targeted perturbations of the cell cycle or transcription factor signaling evoke stimulus-specific changes in accessibility variance, demonstrating that variability is not solely dependent on the cell cycle. The study also reveals that trans-factors promote cell-type-specific chromatin accessibility variation genome-wide and that variation in chromatin accessibility in cis is highly correlated with previously reported chromosome compartments, suggesting a connection to higher-order chromosome folding. Overall, the findings provide new hypotheses about the regulatory mechanisms underlying single-cell heterogeneity.