GSM7467332: 104266-001-041; Mus musculus; RNA-Seq
Hippocampus gene expression and pathway analysis.   The left hemispheres of the brains were snap frozen in liquid nitrogen. The hippocampi were isolated and used to prepare homogenates using glass beads and ribonucleic nucleic acid (RNA) was extracted as described (Salic et al., 2019, doi: 10.3390/ijms20184359). RNA integrity and concentration were examined for each sample using the RNA 6000 Nano LabChip kit and a bioanalyzer 2100 (both Agilent Technologies, Amstelveen, the Netherlands) and the samples were sequenced by GenomeScan BV (Leiden, the Netherlands). RNA sequencing and RNA counts processing were performed as reported previously (Gart et al., 2021; Seidel et al., 2022). Differentially expressed genes were determined using the Deseq2-pipeline (Love et al., 2014, doi: 10.1186/s13059-014-0550-8) with a statistical cut-off of p-value (p) < 0.05 and used for gene enrichment analysis across pathways and biological processes using the Ingenuity Pathway Analysis suite (IPA; www.ingenuity.com, accessed on 15 September 2022).  The upstream regulator analysis tool of IPA was used to assess the activity of upstream regulators as detailed in (Salic et al., 2019). Next Generation Sequencing was performed at Genome Scan (Leiden, the Netherlands). For this, strand-specific cDNA libraries were generated from total-RNA using oligo-dT magnetic beads, mRNA fragmentation, NEBNext Ultra II Directional RNA Library Prep Kit from Illumina, NEB #E7760S/L resulting in 300–500 bp amplified libraries/sample. Libraries were multiplexed, clustered, and sequenced on a NovaSeq6000 v1.5 system (Illumina, New England Biolabs, Inc. Ipswich, MA, USA), with a paired-end, 150 bp sequencing protocol, 29-60 million reads per sample and indexing. Clustering and DNA sequencing using the NovaSeq6000 v1.5 was performed according to manufacturer's protocols. A concentration of 1.1 nM of DNA was used yielding paired end reads (2x 150bp). NovaSeq control software NCS v1.8 was used. Image analysis, base calling, and quality check was performed with the Illumina data analysis pipeline RTA3.4.4 and Bcl2fastq v2.20.