PROJECT SUMMARY The brain is the most complex organ in the body, consisting of hundreds of molecularly, physiologically, and anatomically distinct cells. Recently, methods have been developed that can cost-effectively measure mRNA abundance in tens of thousands of single cells, and this has led to a revolution in the identification and classification of new types of cells in the brain. But these methods only measure one aspect of gene regulation – mRNA levels. To fully understand the transcriptional networks that function in the brain, it will be important to also map the genome-wide binding locations of transcription factors and chromatin modifiers in the myriad of different cell types present in the brain. We propose to develop a method to simultaneously map transcription factor binding and measure mRNA abundance from single cells in the brain. To do so, we will adapt our transposon based methods for measuring the binding of DNA interacting proteins to existing single cell profiling methods. This technology, single-cell Calling Cards, builds on our previously developed transposon Calling Card method, but significantly extends the method allowing use in populations of heterogeneous cells, without a priori definition of cell type. We propose here to develop new mouse lines compatible with the wide range of existing resources in mouse, as well as viral and plasmid reagents applicable across model species, and distribute these to the community. Finally, since the tools we will develop generate new types of data, we will develop a user- friendly software for data visualization and analysis of TFs or chromatin modifying proteins binding data across multiple cell types. Robust resources for analysis are crucial if these technologies are to find broad use in the community. Our primary goal is to enable the parallel analysis of transcription factor binding and mRNA expression levels from tens of thousands of single cells in the brain. !