Cell 179, 772–786 (2019).

Neuroanatomical methods are crucial to understanding how diverse neurons are organized. In MAPseq, a previously developed multiplexed analysis of projections by sequencing, individual neurons are labeled by random RNA barcodes that are used to reveal the projection pattern by matching target areas and source areas. However, MAPseq requires tissue homogenization for sample preparation, which discards the spatial organization of neurons. To map the somatic origin at cellular resolution, Chen et al. developed BARseq (barcoded anatomy resolved by sequencing), which employs in situ barcode sequencing at the source area and microdissection and sequencing at the target projection areas. The combination of transcriptomic and anatomical profiles can relate projection to gene expression when using marker-based Cre-labeled animals. In the mouse auditory cortex, BARseq confirmed the laminar organization of the three classes of projection neurons: intratelencephalic, pyramidal tract-like and corticothalamic. In combination with FISH, BARseq was able to map the projections of transcriptionally defined intratelencephalic subtypes in the mouse auditory cortex.