Gene expression analysis

RNA was extracted from the brain tissue using the ReliaPrep™ RNA Miniprep Systems (Promega, Z6111). RNA integrity numbers were then measured to confirm that the RNA was of high quality. Hybridization was performed using the Gene Kit WT PLUS Reagent Kit (Affymetrix, 902280), and microarray analysis was performed using the Clariom S mouse Assay (Applied Biosystems, 902931). The data were acquired using the GeneChip microarray analysis system GCS 3000Dx (Affymetrix). Microarray data were quality assessed and normalized using Transcriptome Analysis Console (TAC) software (Thermo Fisher); Thermo Fisher array data were normalized using robust multichip analysis (RMA). The data were filtered to include only processed signals within 90% of the overall mean. Normalized data were analyzed using Sabio Platform (https://www.subioplatform.com/ja/). DEGs between the two groups were included in subsequent analyses if the fold change factor was > 1.7-fold and the p-value was < 0.05. The p-value was calculated using Welch’s t test. Additionally, false discovery rate (FDR) calculations to correct for multiple testing were done according to Benjamini. Additionally, G × E interactions were investigated using two-way ANOVA on the signal intensity of each gene. Genes with a statistically significant gene-environment interaction (P < 0.05) were identified as DEGs with G × E interactions and utilized accordingly. GO and pathway analyses were performed using the above data sets, and DAVID (https://david.ncifcrf.gov), GSEA (https://www.gsea-msigdb.org/gsea/index.jsp), and Metascape (https://metascape.org/gp/index.html#/main/step1) were used^32–35. A two-way ANOVA was performed when a dominant interaction between the genotype and environmental factor was observed. When a predominant interaction was observed between the genotype and environmental factor, post-hoc Tukey's multiple comparison test was used to analyze differences between groups differing by only one factor. Statistical analysis and data visualization were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) and Python (https://www.python.org/).