Protocol for mapping neural circuit connectivity with START: Single transcriptome assisted rabies tracing

Characterizing neural connectivity at transcriptomic cell-type resolution is essential for understanding neural mechanisms of circuit function. Here, we present single transcriptome assisted rabies tracing (START), a protocol combining monosynaptic rabies tracing with single-nucleus RNA sequencing to identify transcriptomic cell types providing inputs to defined neuronal populations in the mouse cortex. We describe steps for Cre-dependent helper virus injection, EnvA-pseudotyped rabies infection, tissue microdissection, and fluorescence-activated nuclei sorting. We then detail procedures for library preparation and computational annotation of nuclei. For complete details on the use and execution of this protocol, please refer to Patiño et al.¹.