Protocol to produce and apply barcoded rabies virus for single-neuron input mapping in mice

Barcoded rabies viral tracing enables high-throughput mapping of brain-wide inputs to individual neurons while allowing integration with transcriptomic profiling. Here, we present a protocol to produce and apply barcoded rabies virus in mice for single-neuron input mapping. We describe steps to generate barcode plasmid libraries, produce barcoded rabies virus, and isolate target brain regions. We then detail procedures for preparing single-cell suspensions or bulk RNA, building barcode amplicon libraries from input regions, and performing initial analysis of the sequencing data. For complete details on the use and execution of this protocol, please refer to Tan et al.^1,2.