Projection-TAGs enable multiplex projection tracing and multi-modal profiling of projection neurons.

Single-cell multiomic techniques have sparked immense interest in developing a comprehensive multi-modal map of diverse neuronal cell types and their brain wide projections. However, investigating the spatial organization, transcriptional and epigenetic landscapes of brain wide projection neurons is hampered by the lack of efficient and easily adoptable tools. Here we introduce Projection-TAGs, a retrograde AAV platform that allows multiplex tagging of projection neurons using RNA barcodes. By using Projection-TAGs, we performed multiplex projection tracing of the mouse cortex and high-throughput single-cell profiling of the transcriptional and epigenetic landscapes of the cortical projection neurons. Projection-TAGs can be leveraged to obtain a snapshot of activity-dependent recruitment of distinct projection neurons and their molecular features in the context of a specific stimulus. Given its flexibility, usability, and compatibility, we envision that Projection-TAGs can be readily applied to build a comprehensive multi-modal map of brain neuronal cell types and their projections.