Connectome-seq: High-throughput Mapping of Neuronal Connectivity at Single-Synapse Resolution via Barcode Sequencing.

Understanding neuronal connectivity at single-cell resolution remains a fundamental challenge in neuroscience, with current methods particularly limited in mapping long-distance circuits and preserving cell type information. Here we present Connectome-seq, a high-throughput method that combines engineered synaptic proteins, RNA barcoding, and parallel single-nucleus and single-synaptosome sequencing to map neuronal connectivity at single-synapse resolution. This AAV-based approach enables simultaneous capture of both synaptic connections and molecular identities of connected neurons. We validated this approach in the mouse pontocerebellar circuit, identifying both established projections and potentially novel synaptic partnerships. Through integrated analysis of connectivity and gene expression, we identified molecular markers enriched in connected neurons, suggesting potential determinants of circuit organization. By enabling systematic mapping of neuronal connectivity across brain regions with single-cell precision and gene expression information, Connectome-seq provides a scalable platform for comprehensive circuit analysis across different experimental conditions and biological states. This advance in connectivity mapping methodology opens new possibilities for understanding circuit organization in complex mammalian brains.