gene sets upon neuronal activation following NEAT1 knockdown. We next investigated whether the genes in these enriched gene sets were significantly up or down-regulated in our experimental conditions. First, for the control condition, GSEA performed on 5,339 significantly altered genes from mock-treated control iPSC-derived neurons following activation (Supplementary Fig. 7a; Control ASO KCl versus Control ASO) showed that NEAT1 expression is correlated with decreased ion channel gene set expression (Fig. 3b) implying a normal role in decreasing ion channel excitation. Second, to demonstrate that this correlation arises from a direct role of NEAT1 in regulating ion channel gene expression, we showed that NEAT1 knockdown in activated neurons (Supplementary Fig. 7a; Neat1 ASO KCl versus Control ASO KCl) is itself sufficient to drive a significant increase in ion channel gene set expression relative to controls (Fig. 3c) demonstrating that NEAT1 is necessary for the proper modulation of neuronal excitability. Gene ontology (GO) analysis of genes differentially expressed in activated NEAT1-knockdown neurons relative to activated controls further revealed an enrichment for apoptosis, cell death and stress related pathways, suggesting that reduction of NEAT1 may induce a neuronal stress response (Supplementary Figs 9 and 10).
