Summary
Understanding neural circuits requires deciphering the interactions of myriad cell types defined by connectivity, spatial organization, gene expression, and other properties. Resolving these cell types requires both single neuron resolution and high throughput, a combination that is challenging to achieve with conventional anatomical methods. Here we introduce BARseq, a method for mapping the projections of thousands of spatially resolved neurons by RNA barcoding. We used BARseq to determine the projections of 1309 neurons in mouse auditory cortex to 11 targets. We observed 264 distinct projection patterns. Hierarchical clustering confirmed the major classical classes of projection neurons, as well as further subdivisions within each class. We further combined BARseq with in situ detection of mRNA to relate neuronal gene expression with projections. By bridging high-throughput neuroanatomy with high spatial resolution, BARseq provides a path towards a systematic multi-modal description of the nervous system.