Figure 6.

Validation of RNA-Seq results by qRT-PCR and in situ hybridization. A, qRT-PCR validation of cell type-enriched genes identified by RNA-Seq. We performed qRT-PCR with Fluidigm BioMark microfluidic technology. Expression of several genes identified by RNA-Seq as enriched in each cell type was examined by qRT-PCR. The housekeeping gene Gapdh was included for comparison. Twelve replicates of each purified cell type and three replicates of whole-brain samples were analyzed. Warmer colors represent lower Ct values (higher abundance of transcripts), and cooler colors represent higher Ct values (lower abundance of transcripts). Black indicates no amplification. Data of genes labeled in red were quantified in B. B, Ct differences of Atp13a4, Cpne7, Fam70b, Tmem88b, and Rcsd1 compared with Gapdh were plotted on a log2 scale. Error bar represents SD. RNA-Seq analysis showed that Atp13a4, Cpne7, Fam70b, Tmem88b, and Rcsd1 are enriched in astrocytes, neurons, OPCs, oligodendrocytes, and microglia, respectively. qRT-PCR validated these results. C–G, In situ hybridization validated novel cell type-specific genes. Left, Low-magnification image of the cortex and hippocampus. Scale bar, 200 μm. Right, High-magnification image of the cortex. Scale bar, 50 μm. Fluorescence in situ hybridization signals with probes against novel cell type-specific genes (red) and known cell type-specific markers (green) are shown. The regions in the yellow boxes are enlarged and shown as single channel and merged images on the right.