Spatial mapping of RNAs using Expansion Sequencing (ExSeq). a Targeted ExSeq library preparation: (i) RNA anchoring and expansion, (ii) padlock probe hybridization, (iii) probe ligation, and (iv) rolling circle amplification. b Amplicon counts for targeted ExSeq versus HCRv3.0-amplified ExFISH for the same transcript in the same HeLa cell (60 cells). c Targeted ExSeq of 42 cell type marker genes in Thy1-YFP mouse visual cortex. (Top) Fluorescence image showing targeted ExSeq reads (red) and YFP (green). (Bottom) Localization of marker genes Pvalb (red), Sez6 (cyan), Slc32a1 (magenta), and Gad2 (yellow) with YFP (green). Scale bar, 20 μm. d Targeted ExSeq gene expression profiles of 1154 cells clustered into 15 cell types. e Spatial organization of cell types identified in d. Cell-segmented reads are shown, colored by cluster assignment, and overlaid on YFP (white).