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Introduction

The development of spatially resolved genomic assays enables molecular analysis of tissues, with the potential of revealing how single-cell gene activity orchestrates the structure of complex tissues like the nervous system. Here, we use the MERSCOPETM Platform to generate a transcriptionally defined and spatially resolved single-cell mouse brain atlas. By performing multiplexed error-robust fluorescence in situ hybridization (MERFISH) assays with a 500-gene panel designed for cell typing, we obtained over two million cells with precise gene expression and spatial information across the mouse brain. Clustering analysis of the gene expression data resolved all major cell populations as well as detailed neuron and nonneuron subtypes across different brain regions. By assessing the relationship between molecular and anatomic features of identified cell types, we found that both excitatory and inhibitory neuron subtypes exhibit significant variation in gene expression and spatial distribution along multiple axes of different brain structures. Furthermore, the high-resolution spatial transcriptomic data enabled us to assess the spatial relationship and cell-cell interactions across different cell types. Altogether, our work not only created a molecularly defined and spatially resolved mouse brain cell atlas, but also demonstrated the power of MERFISH measurements generated by the MERSCOPE Platform in analyzing the molecular basis underlying the anatomic and functional complexity of the nervous system.