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

Single cell genomics has revolutionized our understanding of the diversity 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 strategy to generate high-complexity barcoded rabies virus (RV) for scalable circuit mapping that is compatible with both single-cell and single-nucleus RNA sequencing (sc/snRNA-seq) readout. Our barcoded RV libraries contain up to 43 million unique barcodes with a relatively uniform distribution, allowing multiplexed circuit mapping from tens of thousands of individual starter cells. We demonstrate the utility of this approach by mapping the emerging circuits in the developing human cortex using organotypic slice cultures. 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. Overall design: Sequencing of barcoded G-deleted rabies (RVdG) libraries for quantitative analysis