Sample preparation for microarray hybridization was carried out as described in the NuGEN Ovation PicoSL WTA System V2 and NuGEN Encore Biotin Module manuals (NuGEN Technologies Inc., San Carlos, CA, USA). Briefly, 20 ng of total RNA was reverse-transcribed into double-stranded cDNA in a two-step process, introducing a SPIA tag sequence. Bead-purified cDNA was amplified by a SPIA amplification reaction, followed by an additional bead purification. Four micrograms of SPIA cDNA was fragmented, terminally biotin-labeled, and hybridized to Affymetrix Mouse Gene 2.0 ST arrays for 16 hours at 45°C in a GeneChip Hybridization Oven 640. Hybridized arrays were washed and stained in an Affymetrix Fluidics Station FS450, and the fluorescent signals were measured with an Affymetrix GeneChip Scanner 3000 7G. Fluidics and scan functions were controlled by the Affymetrix GeneChip Command Console v4.1.3 software. Sample processing was performed at an Affymetrix Service Provider and Core Facility, “KFB—Center of Excellence for Fluorescent Bioanalytics” (Regensburg, Germany; www.kfb-regensburg.de). Raw CEL files were normalized using robust multi-array average algorithm from “affy” R package (https://bioconductor.org/packages/release/bioc/html/affy.html) and annotated using pd.mogene.2.0.st from “Oligo” R package (https://bioconductor.org/packages/release/bioc/html/oligo.html). Student’s t test P values, adjusted P values (BH test), and log2 fold changes were calculated using “EMA” R package (https://cran.r-project.org/web/packages/EMA/index.html). Probes that were not annotated with National Center for Biotechnology Information gene ID or annotated as predicted genes were not included in subsequent analysis.

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