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 complex wiring diagram, 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. Projection neurons in the mouse cortex are labeled with Projection-TAGs. Nuclei are extracted and isolated using FACS based on 7-AAD (all nuclei) or 7-AAD and GFP (Projection-TAG+ nuclei). Sorted nuclei are then sequenced using 10X Genomics Chromium Single Cell Gene Expression 3’ V3.1 Assay or 10X Genomics Chromium Single Cell Multiome Assay.