Understanding the function of a tissue requires knowing the spatial organization of its constituent cell types. In the cerebral cortex, single-cell RNA sequencing (scRNA-seq) has revealed the genome-wide expression patterns that define its many, closely related cell types, but cannot reveal their spatial arrangement. Here we introduce probabilistic cell typing by in situ sequencing (pciSeq), an approach that leverages prior scRNA-seq classification to identify cell types using multiplexed in situ RNA detection. We applied this method to map the inhibitory neurons of hippocampal area CA1, a cell system critical for memory function, for which ground truth is available from extensive prior work identifying the laminar organization of subtly differing cell types. Our method confidently identified 16 interneuron classes, in a spatial arrangement closely matching ground truth. This method will allow identifying the spatial organization of fine cell types across the brain and other tissues.

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