Transcriptional characterization of excitatory layer 4 (L4) neurons receiving intact whisker inputs. A, UMAP of 76,232 reclustered excitatory neuronal nuclei annotated by cortical layer identity. B, Feature plots for neuronal marker Map2, excitatory cell marker Slc17a7 and Layer 4 marker Rorb expression used to define L4 excitatory neurons. C, mFISH validation of L4 neurons in S1BC showing coexpression of Map2, Slc17a7, and Rorb, counter-stained with DAPI. Left, Composite image of S1BC with boxed area expanded at right to show cortical layers in split channels. Bottom left, Image showing L4 excitatory nuclei positive (white arrows) and negative (red arrows) for all three markers. Scale bars: 200 µm (top left), 10 µm (bottom left), 100 µm (right panels). D, Volcano plot with differentially expressed genes (DEGs) in L4 excitatory neurons comparing sham and ION-X mice. E, Gene Ontology (GO) enrichment analysis of upregulated biological processes, ranked by q value (adjusted p value), colored by number of DEGs. See also Dataset S3.