WGCNA identifies co-expression modules associated with neuronal differentiation and depolarization. (A) Flowchart illustrating the experimental procedures and expression analysis. (B) Hierarchical clustering of genes based on gene co-expression pattern across progenitors, neurons at rest, and after depolarization. Identified co-expression modules were represented by color classifiers, noted across the top of the dendrogram. The `differentiation’, `depolarization’, and the `TSmutation’ color bars represent the correlation values between gene expression and three biological traits: differentiation, depolarization, and CACNA1C G406R mutation status, respectively. Red signifies upregulation, while blue signifies downregulation. Only the genes with a trait correlation larger than 0.5 or smaller than -0.5 are marked in the plot. (C-I) Module eigengene patterns and enrichment scores of the top five enriched GO categories for module (C) turquoise, (D) blue, (E) brown, (F) tan, (G) yellow, (H) midnightblue, and (I) green. Samples are ordered by control progenitors (CP), patient progenitors (TP), control resting neurons (CN), patient resting neurons (TP), control depolarized neurons (CN+), and patient depolarized neurons, as illustrated by the key at the bottom. (J) Module-level enrichment for previously identified differentiation- and depolarization-associated gene sets curated from: (1) Kang et al. developing postmortem human brain ,; (2) Stein et al. phNPC expression study (phNPC_wk1vswk8_up/down) ; (3) McKee et al. ; and (4) Kim et al.  KCl-induced depolarization expression studies. `brain_s1vss8_up/down’ represent the genes that are either up- or downregulated between stage 1 vs. stage 8, while `phNPC_wk1vswk8_up/down’ are the group of the genes up- or downregulated between week 1 vs. week 8 differentiation, as defined in the paper ,. Cells are colored to reflect enrichment significance with ceiling of 10-4. Enrichment odds ratios are shown in the table if the P <0.05 (*FDR <0.05).