Fig. 1: Spatial maps of molecular cell types across the adult mouse CNS at subcellular resolution.

a, Overview of the study. Mouse brain tissue slices were collected four to five weeks after systemic administration of barcoded AAVs. STARmap PLUS^3,4 was performed to detect single RNA molecules from a targeted list of 1,022 endogenous genes and the trans-expressed AAV barcodes. The RNA spot matrix was converted to a cell-by-gene expression matrix via ClusterMap¹². By integrating with existing mouse CNS scRNA-seq data¹, we generated a CNS spatial atlas with cell cluster nomenclatures jointly defined by molecular cell types and molecular tissue regions, and imputed single-cell transcriptome-wide expression profiles. circRNA, circular RNA; AC, astrocytes; CB, cerebellum; CHO/MA, cholinergic and monoaminergic neurons; CHOR, choroid plexus epithelial cells; DE/MEGLU, diencephalon/mesencephalon excitatory neurons; DE/MEINH, diencephalon/mesencephalon inhibitory neurons; DGGRC, dentate gyrus granule cells; EPEN, ependymal cells; GNBL, glutamatergic neuroblasts; HB/SP, hindbrain/spinal cord; HYPEN, subcommissural organ hypendymal cells; MGL, microglia; MSN, telencephalon projecting inhibitory neurons (or medium spiny neurons); NGNBL, non-glutamatergic neuroblasts; OBINH, olfactory inhibitory neurons; OEC, olfactory ensheathing cells; OLG, oligodendrocytes; OPC, oligodendrocyte precursor cells; PEP, peptidergic neurons; PER, pericytes; PVM, perivascular macrophages; TEGLU, telencephalon projecting excitatory neurons; TEINH, telencephalon inhibitory interneurons; VEN, vascular endothelial cells; VLM, vascular and leptomeningeal cells; VSM, vascular smooth muscle cells. b, Uniform manifold approximation and projection⁵⁰ (UMAP) of 1.09 million cells coloured by subcluster. The surrounding diagrams show 230 subclusters from 26 main clusters. Top right, UMAP coloured by slice directions; bottom right, UMAP coloured by slice identity as in c. c, Molecular cell-type maps of the 20 mouse CNS slices coloured by subcluster. Each dot represents one cell. d, A zoomed-in view of tissue slice 12 in c. Each dot represents a DNA amplicon generated from an RNA molecule, colour-coded by its cell-type identity. Brain region abbreviations are based on the Allen Mouse Brain Reference Atlas^18,19,20. alv, alveus; cc, corpus callosum; chpl, choroid plexus; cing, cingulum bundle; CP, caudoputamen; CTX, cerebral cortex; df, dorsal fornix; DG, dentate gyrus; FC, fasciola cinereum; fi, fimbria of hippocampus; HPF, hippocampal formation; int, internal capsule; L2/3, layer 2/3; L4, layer 4; L5, layer 5; L6, layer 6; LH, lateral habenula; MH, medial habenula; mo, molecular layer; po, polymorph layer; RSP, retrosplenial cortex; RT, reticular nucleus of the thalamus; sg, granule cell layer; slm, stratum lacunosum-moleculare; so, stratum oriens; sp, pyramidal layer; sr, stratum radiatum; STR, striatum; TH, thalamus; v3, third ventricle; VL, lateral ventricle. e, A zoomed-in view of the habenula region in d with cell boundaries outlined (left) and a mesh graph of physically neighbouring cells connected by edges (middle). Symbols for cell types with more than two counts were labelled (right). HABCHO, habenular cholinergic neurons; HABGLU, habenular excitatory neurons; INH, inhibitory neurons; NA, unannotated (see Methods, ‘Main cluster and subcluster cell-type annotation’). f, A representative fluorescent image of the region highlighted in e from the first cycle of SEDAL. Each dot represents an amplicon.