Virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq) defines projection neurons involved in sensorimotor integration

Behavior arises from concerted activity throughout the brain. Consequently, a major focus of modern neuroscience is defining the physiology and behavioral roles of projection neurons linking different brain areas. Single-cell RNA sequencing has facilitated these efforts by revealing molecular determinants of cellular physiology and markers that enable genetically targeted perturbations such as optogenetics, but existing methods for sequencing defined projection populations are low throughput, painstaking, and costly. We developed a straightforward, multiplexed approach, virally encoded connectivity transgenic overlay RNA sequencing (VECTORseq). VECTORseq repurposes commercial retrogradely infecting viruses typically used to express functional transgenes (e.g., recombinases and fluorescent proteins) by treating viral transgene mRNA as barcodes within single-cell datasets. VECTORseq is compatible with different viral families, resolves multiple populations with different projection targets in one sequencing run, and identifies cortical and subcortical excitatory and inhibitory projection populations. Our study provides a roadmap for high-throughput identification of neuronal subtypes based on connectivity. [Display omitted] •Retrograde viruses express mRNA at levels detectable in single-cell sequencing•Different transgenes can be multiplexed in a single sequencing run•VECTORseq identifies both cortical and subcortical projection neurons•VECTORseq defines new superior colliculus and zona incerta projection populations Neurons with different axonal projection targets and distinct molecular and functional properties are intermingled throughout the brain. Cheung et al. establish a high-throughput method, VECTORseq, to transcriptionally define projection neurons. The method reimagines transgenes expressed by widely used retrogradely infecting viruses as multiplexed RNA barcodes that are detected in single-cell sequencing.