RNA sequencing
RNA sequencing was performed as previously described (Baik et al., 2021). Brain hemispheres were snap frozen in liquid nitrogen. In a separate cohort of mice, the hippocampus was dissected from the brain and snap frozen in liquid nitrogen. Brain hemispheres and hippocampi were sonicated in TRIzolTM (Life Technologies, USA), mixed with chloroform, and centrifuged at 824 x g for 15 min at 4 °C. The aqueous phase was collected and RNA was extracted using the RNeasy® Micro Kit (Qiagen, USA). RNA was quantified using a NanoDrop One spectrophotometer (Thermo Scientific, USA) and then stored at -80°C. The RNA samples were shipped to NovogeneAIT Genomics (Singapore) for cDNA library preparation and RNA sequencing. mRNA was purified from total RNA using poly-T oligo-attached magnetics. mRNA was converted to cDNA and purified using AMPure XP Beads (Beckman Coulter Life Sciences, USA). cDNA libraries were acquired by PCR amplification. High-throughput sequencing was conducted using the HiSeqTM2500 platform (Illumina, USA). The results were mapped to the Ensembl-released mouse genome sequence and annotation. Differential expression analysis was conducted using the DESeq R Package V.1.10.1 and P-values were adjusted using the Banjamini and Hochberg’s approach for controlling the false discovery rate. Genes were considered differentially expressed if the adjusted P-value was less than 0.05. R package heatmap3 and log2Fold-Change output from EdgeR V.3.2.4 were used to create heatmaps for differentially expressed genes.