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.