MACS-FACS-seq of Layer 5 cortical neurons after subcortical stroke. Schematic representation of workflow to isolate stroke-injured and neighboring uninjured CTIP2+ Layer 5 cortical neurons for RNA-sequencing (a). Retrograde neuronal tracing with fluororuby (FR) within subcortical white matter at the stroke site (white arrow, upper panel, b) and deep layers of overlying cortex (yellow arrow, upper panel, b) 7 days after stroke. CTIP2+ (green) and FR+ (red) Layer 5 cortical neurons overlying stroke (lower panels, b). Enrichment for Layer 5-specific marker genes in MACS-FACS isolated CTIP2+ cortical neurons (average fkpm; n = 5; p < 0.0001 by 2-way ANOVA) (c). Volcano plot of mapped gene sequences between CTIP2+/FR- and CTIP2+/FR+ cortical neurons (d). Gene ontology of differentially expressed genes (FDR < 0.1) (e). Dashed line indicates p = 0.05. Scale bars = 500 μm in upper panel and 10 μm in lower panels of b.