Supplementary Figure 3: Quantitative PCR supports altered gene expression identified by RNA-seq and hierarchical clustering of genes differentially expressed in several top canonical pathways.

(a-f) New differentiations of representative non-disease (21Q) and HD (109Q) lines were performed and select genes evaluated by RT-qPCR. All exhibited altered expression consistent with that identified by RNA-seq. Two differentiated wells of each line were pooled for RNA extraction and three technical replicates of qPCR performed. A statistical difference in gene expression was determined using an unpaired two-tailed t-test in GraphPad Prism. NEUROD1: P=0.0003, t=11.38, df=4; CALB1: P=0.0005, t=10.23, df=4; CAMK4: P=0.0003, t=11.72, df=4; POU4F2: P=0.0005, t=10.25, df=4; GAD1: P<0.0001, t=21.15; df=4; KCNQ3: P<0.0001, t=24.22, df=4). (g) Heat map showing hierarchical clustering of log2 transformed gene expression values (RPKMs) of the significantly differentially expressed genes (1869) found in several of the top affected canonical pathways (colors displayed by row min and max values, yellow = higher and blue = lower expression). Genes/rows are sorted by involvement in specific pathways that have been marked by color and shows pathway convergence on key regulator genes founds in both axonal guidance and either CREB or WNT signaling. Fold change is shown to the right of the gene symbol and represents the average change of HD/non-disease. A clear pattern of differential gene expression in each pathway provides an indication of activation or inhibition of the pathway. (h) Table showing normalized log2 gene expression values of the top up and down regulated genes in Fig. S3g. Each gene is categorized by pathway involved and HD vs non-disease fold change is displayed.