PROJECT SUMMARY Predicting cis-regulatory information from sequence alone is not yet possible, but collecting cis-regulatory information systematically across all human cell types and across species would be feasible with significant progress in technology. Here we propose to develop such breakthrough technology that will allow to extract cis-regulatory information from any cell type, including mixtures of heterogeneous cell types. The technology is based on a transposase system that combines ChIP and ATAC-seq technology, and thus allows the simultaneous measurement of chromatin accessibility and transcription factor occupancy in the same cells. It is innovative in its orders of magnitude improvement in efficiency by which DNA fragments enter the genomic library, which allows the assay to be performed at single-cell resolution. It is cutting-edge in its resolution, which allows the identification of precise footprints of transcription factor bound in vivo. We will optimize scale and workflow, as well as develop an initial analysis framework, to make the technology applicable to a wide range of systems. As proof-of-principle, we will apply the technology to early mouse embryos and compare the results to those obtained from mouse embryonic stem cells. Having such technology will open the door to unprecedented explorations of cis-regulatory information across any cell type. It will deepen our understanding of transcriptional regulatory networks during development and evolution, and will provide insights into mutations and mechanisms underlying human disease.