PROJECT ABSTRACT To meet the goal of the BRAIN Initiative Cell Census Network to catalogue and produce molecular profiles of every cell type in the human brain; order-of-magnitude improvements in single-cell assay throughput and coverage are required. In this proposal, we detail strategies to meet these needs by leveraging novel chemistry workflows and combinatorial indexing techniques. This will include the development of two complementary techniques (s3 and s4) that overcome the major limitations of current technologies to produce far higher coverage of DNA-based properties, including chromatin accessibility (s3-ATAC) and histone marks (s4-CAT). These technologies will also be leveraged to develop assays that capture RNA transcription alongside the DNA-encoded property. We will also extend these workflows to produce order-of-magnitude improvements in cell coverage for our previously-described technology to profile single-cell DNA methylation in high throughput (sci-MET). The improved coverage will also enable the development of targeted capture for pooled single-cell DNA methylation libraries to reduce sequencing costs per cell while profiling regions of interest. These capture libraries will enable much larger cell datasets that can complement whole-genome single-cell profiles from a smaller sampling of cells. Finally, we will demonstrate these technologies by producing a preliminary atlas of epigenetic profiles in the human primary visual cortex and hippocampus to serve as a starting point for the large scale coordinated efforts of the BICCN.

PROJECT NARRATIVE The ambitious goals of the BRAIN Initiative Cell Census Network (BICCN) to map the molecular profiles of every cell type in the human brain requires the development of technologies with order-of-magnitude improvements in cell coverage and throughput. Here we propose the development of several technologies that will produce high-coverage cell profiles for several epigenetic properties, including the profiling of active regulatory elements and DNA methylation. These techniques are capable of producing hundreds-of-thousands of profiles in a single experiment and will be used to build a preliminary epigenetic atlas in the human visual cortex and hippocampus.