High-Complexity Barcoded Rabies Virus for Scalable Circuit Mapping Using Single-Cell and Single-Nucleus Sequencing

SUMMARY Single cell genomics has revolutionized our understanding of neuronal cell types. However, scalable technologies for probing single-cell connectivity are lacking, and we are just beginning to understand how molecularly defined cell types are organized into functional circuits. Here, we describe a protocol to generate high-complexity barcoded rabies virus (RV) for scalable circuit mapping from tens of thousands of individual starter cells in parallel. In addition, we introduce a strategy for targeting RV-encoded barcode transcripts to the nucleus so that they can be read out using single-nucleus RNA sequencing (snRNA-seq). We apply this tool in organotypic slice cultures of the developing human cerebral cortex, which reveals the emergence of cell type– specific circuit motifs in midgestation. By leveraging the power and throughput of single cell genomics for mapping synaptic connectivity, we chart a path forward for scalable circuit mapping of molecularly-defined cell types in healthy and disease states.