Affymetrix genechip mouse clariom s arrays

For gene expression analysis, tissue samples were obtained from the dorsal hippocampus. Total RNA was extracted from the tissue using the RNeasy Plus Mini Kit (Qiagen Inc., Valencia, CA, USA). Microarray studies were performed using Affymetrix GeneChip Mouse Clariom S arrays (Thermo Fisher Scientific, Hampton, NH, USA). Gene expression analysis was performed using iDEP (integrated Differential Expression and Pathway analysis; Steven, Runan, 2018, BMC Bioinformatics).

Whole hearts were excised and flash frozen in liquid nitrogen. Total RNA was isolated using an RNeasy fibrous tissue kit. Verification of RNA integrity and RNA quantification were done by spectrophotometry and an RNA 6000 Nano assay (Bioanalyzer 2100, Agilent Technologies).
The RNA integrity number for all samples was >6 (mean=8.6 +/-SEM 0.19). Total RNA (250 ng) was primed for the entire length of RNA, including both poly(A) and non-poly(A) mRNA and reverse transcribed to generate sense-strand targets that were biotin-labelled using a WT Plus Reagent kit, and then hybridized to Affymetrix GeneChip Mouse Clariom S arrays for 16 hours (48 o C), following manufacturer's instructions (Thermo Fisher Scientific). Hybridization cocktails were removed, and arrays were washed and stained on a Fluidics Station 450 (mouse Clariom S arrays). Arrays were scanned on the Affymetrix GCS3000 7G scanner and initial quality control data evaluated using Affymetrix Expression Console software (Thermo Fisher Scientific).
Microarray data was imported and analyzed (ANOVA p<0.05, 1.5 fold cutoff, 0.1 false discovery rate) using the Transcriptome Analysis Console (Applied Biosciences). Gene ontology (GO) enrichment analysis was performed with the GOrilla tool using a single rank-ordered gene list 25, 26 .