ABSTRACT – PROJECT 1: UW-CNOF MAPPING TECHNOLOGY DEVELOPMENT Over the past decade, advances in technologies for assaying genome architecture have led to remarkable progress in our understanding of the 4D nucleome, i.e. the spatiotemporal organization of the eukaryotic genomes within nuclei. Among all of the powerful experimental tools that have recently emerged, chromosome conformation capture (3C) and its high-throughput derivatives have become the most widely used methods for characterizing genome architecture both locally and globally. However, the current repertoire of 3C-based methods is crucially limited with respect to key parameters such as specificity, resolution and input requirements. Recently, we have made substantial progress in addressing these limitations with DNase Hi-C, a restriction enzyme-free derivative of the Hi-C protocol. Here, we propose to further develop biochemical methods for characterizing the dynamic 4D nucleome that substantially improve upon the state of the art with respect to input requirements (down to single cell), resolution (eliminating restriction enzyme bias), scale (genome-wide or targeted views) and integration (combined measurements with the transcriptome and epigenome), while also improving sensitivity, specificity, simplicity and throughput. In Aim 1, we will continue to optimize genome-wide and targeted DNase Hi-C protocols – including a much simplified, in situ version of DNase Hi-C – to further minimize input requirements and bias while improving resolution. We will also refine these protocols in order to maximize robustness, scalability and exportability. In Aim 2, we will develop a high- throughput method for routinely measuring genome architecture in large numbers of single cells. Our proposed approach, based on combinatorial indexing and supported by substantial preliminary data, enables the routine production of DNase Hi-C (nuclear architecture) or ATAC-seq (chromatin accessibility) data from hundreds to thousands of single cells per experiment. In Aim 3, we will integrate DNase Hi-C and other assays for the concurrent measurement of genome architecture, epigenetic state, and the transcriptome, in each of many single cells. We believe that the successful development of such co-assays will profoundly advance our ability to develop integrative models connecting genome form and function. Finally, in Aim 4, we will standardize, benchmark, and export the experimental methods developed by this project, with the goal of maximizing their impact and utility for NOFIC investigators, the 4DN Network, and the broader scientific community.