Development and application of a barcoded rabies viral tracing method for mapping brain-wide inputs to single neurons

While whole-brain connectome mapping has been achieved at both macroscopic (tract tracing) and microscopic (electron microscopy) scales, existing methods—such as conventional rabies virus tracing or sequencing-based projection mapping—cannot simultaneously capture both local and long-range inputs to single neurons while integrating transcriptomic identities. Furthermore, prior barcoded rabies approaches faced challenges such as barcode sharing between starter cells, low barcode/cell recovery rates, and an inability to link connectivity to gene expression. The Barcoded Rabies Viral Tracing (BRT) method was engineered to address these gaps by combining high-diversity barcoded rabies viruses with single-cell RNA sequencing of the starter region and bulk barcode sequencing of input regions, enabling the reconstruction of comprehensive inputomes for transcriptome-defined neurons.