Fig 5. Quantitative RNA sequencing to identify differentially expressed genes in PAs versus non-PAs isolated from Mlc1-EGFP;GLAST-DsRed adult mice.

(A); Strategy for dissection and analysis of cells from cerebral cortices of Mlc1-EGFP;GLAST-DsRed adult mice. Non-PAs (DsRed⁺ cells) and PAs (EGFP⁺/DsRed⁺ cells) were fractionated using FAC-based sorting approaches followed by comparative RNA sequencing. (B); Flow cytometry plot showing the profile of sorted DsRed⁺ single positive cells (non-PAs) and EGFP⁺/DsRed⁺ double positive cells (PAs) from the cerebral cortices. Note that the majority of cells are DsRed⁺ and nearly all EGFP⁺ cells are double positive for DsRed. The few cells in the lower box are EGFP⁺ single positive PAs that lack DsRed expression. (C, D); Quantitative RNA sequencing comparisons reveal differentially expressed genes in PAs versus non-PAs as revealed by a color-coded heat map (C) and a volcano plot (D). Red indicates higher mRNA expression levels and blue indicates lower expression in PAs versus non-PAs. Differentially expressed genes were identified using the EdgeR package with adjusted p-value cutoffs <0.05 and log2 fold changes > 2. (E) Independent validation of differential gene expression using mRNA isolated from PAs (EGFP⁺/DsRed⁺) and non-PAs (EGFP^-/DsRed⁺). Note that the select genes analyzed show enrichment in PAs as compared to non-PAs, as revealed by qRT-PCR. Error bars indicate SE of the mean, n = 3. The red bars indicate EGFP^-/DsRed⁺ cell fractions. Expression differences between sorted cells were determined using two-way analysis of variance with Bonferroni post-hoc test, *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001.