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Characterizing the transcriptomic profiles of individual cells by single-cell RNA sequencing (scRNA-seq) has become a universal tool to identify both known and novel cell types and to understand tissue structure and function, ushering in a new era of single cell biology. This has proven to be especially true in complex organs with high cellular heterogeneity, such as the mammalian brain. However, scRNA-seq utilizes dissociated cells and results in the loss of spatial organization of the cell population being analyzed. Validation and spatial mapping of scRNA-seq results can be obtained using assays that retain spatial organization, such as RNA in situ hybridization (ISH). We validated the major gene signatures identified by scRNAseq, including discrete D1 and D2 medium spiny neuron (MSN) subtypes: Drd1a/Foxp1, Drd1a/Pcdh8, Drd2/Htr7, and Drd2/Synpr. Further cellular heterogeneity within the MSN subpopulations was marked by a transcriptional gradient, which we could spatially resolve with RNA ISH, revealing that cells highly expressing one end of the gradient were located in a region adjacent to cells highly expressing the other end of the gradient, with a small overlapping region containing co-expressing cells. Lastly, we validated heterogeneity within non-neuronal striatal cell types, including vascular smooth muscle cells, endothelial cells, microglia, macrophages, and oligodendrocytes.
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