DCX-DsRed+ cells form a distinct, primary population but contains 4 subpopulations with distinct expression patterns. (A) Enrichment of DCX-DsRed⁺ cells through FACS. Sample fluorescence-side scatter dot plot graphs of dissociated single DG cells from a DCX-DsRed transgenic mouse (left) and a wild-type control mouse (right). Both DsRed High (red) and DsRed Low (white) populations in the transgenic mouse are absent from control mouse. We selected the DsRed-high population for subsequent analysis. (B) A schematic of the experimental design for single-cell transcriptome analysis. DG tissue was dissected from mice and dissociated into single cells, which were then subjected to FACS enrichment. DCX-DsRed⁺ cells were then loaded into the Fluidigm C1 system, which captures single cells, isolates RNA, and prepares cDNA within the capture chamber. Sequencing libraries were prepared outside of the C1 machine and RNA-sequencing was performed as described in Materials and Methods. Downstream analyses were carried out on expression data, and discoveries were validated histologically in brain sections. (C) Unsupervised clustering of whole-transcriptome RNA-seq data from individual DCX-DsRed⁺ cells (light blue) alongside individual Nestin-CFP⁺ cells (green, published by Shin et al (2015)) and whole DG tissue (dark blue). (D) PCA analysis of the same data. (E) Distance heatmap of the same data shows that DCX-DsRed⁺ cells tend to be more similar to DG tissue than Nestin-CFP⁺ cells. (F) Expression heatmap and clustering of cell samples and genes using the most variable 400 genes according to ANOVA significance. Each column represents a cell, and each row represents a gene. Red color reflects high expression, and blue represents low expression. (G) PCA plot of the DCX⁺ cell samples based on the 400 most variable genes. Samples are color-coded to match their group membership as shown in Figure 3A. (H) Loading PCA plot of genes used in Figure 3B. Genes are color-coded to match their group membership as shown in Figure 3A. (I) PCA analysis was repeated on cell samples using the entire transcriptome to confirm that DCX-DsRed⁺ subpopulations segregate according to their full expression profiles. (J) Sample group membership of cells when clustered by the whole transcriptome (top) and the most variable 400 genes (bottom). Colored lines show the relative position of each cell sample that belonged to the same group in both clustering methods, and black dotted lines indicate samples that were clustered into different groups.