Fig. 7.

Validation of select cell type specific DEGs using RNAscope in situ hybridization. Representative fluorescent microphotographs for Sham and TBI showing each DEG of interest (selected from Fig. 6a) along with cell marker genes with DAPI in the background. The RNAscope images for Sham (first column) and TBI (third column) display cell colocalization of each DEG (green) and the corresponding cell marker gene (red). The corresponding FISH-quant images for Sham (second column) and TBI (fourth column) show the automated cell segmentation (blue) overlayed on the 2D maximal projection of the DAPI z-stack images (gray) with the DEG of interest (green in the RNAscope images) indicated in bright dots. The quantification of the cell type specific DEGs is shown in the violin plots (5th column) with Sham in blue and TBI in red. Only cells which meet a certain count threshold for the cell type marker gene are considered the appropriate cell type and used in the DEG quantification (details in Methods). The ln(counts per cell) of the DEGs are shown on the y-axis with the p-value from a bimodal likelihood ratio test and log fold change (logFC) between TBI and Sham samples indicated. These figures have been zoomed in 5x from the original form to show high magnification detail of a few cells to more easily demonstrate colocalization of DEGs and cell markers. Lower magnification photos which indicate the region which was magnified are provided in Supplementary Fig. 11. Scale bars are 4 µm. Sample size was n = 8 mice/group