Mapping brain-wide monosynaptic inputs to single neurons with ROInet-seq

Viral projection tracing strategies help build regional connectomes of mammalian brains. G-deleted rabies virus (ΔG-RV) establishes monosynaptic input connectivity but cannot distinguish networks at cell resolution. Here, we implement a barcoded ΔG-RV for network tracing by quantitative RNA-sequencing. At optimized library complexity and uniformity, barcode detection reliably distinguished individual monosynaptic input networks of multiple infected neurons in parallel. To scale this approach to hundreds of cells and full-brain inputs, we develop regions-of-interest network sequencing (ROInet-seq)-an accessible, scalable, and low-cost spatial assay. ROInet-seq combines routine fluorescent imaging of fixed tissue sections with targeted barcode sequencing, enabling brain-wide mapping of single-neuron networks. In the somatosensory cortex, the assay reveals dominant regional contributions that inputs from a handful of thalamic neurons diverged to multiple neurons and frequent convergent ipsilateral and contralateral cortical inputs. Integration with commercial spatial transcriptomics assays improves resolution, together establishing a scalable framework to resolve single-cell network architectures.