2.5. Quantitative RT-PCR and RNA-Seq

RNA was extracted with RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. RNA concentration and purity were evaluated with Nanodrop 1000 (Thermo Scientific, Belgium). RNA (0.25 µg) was converted to cDNA using high-capacity cDNA reverse transcription kit (Applied Biosystems). Quantitative RT-PCR was performed with 6.25 or 0.125 ng cDNA using predesigned primers (IDT) and TaqMan Universal PCR Master Mix (Applied Biosystems), respectively. RNA-Seq expression profiling was performed by the Genomics Core UZ Leuven. Per independent experiment RNA from three technical replicates per experimental condition was pooled and 3 µg RNA/experimental condition were sequenced from a total of three independent experiments. Illumina TruSeq stranded mRNA kit was used and the single-end sequencing was performed. 33 M 50 bp reads per sample were sequenced. Reads were aligned to mm10 murine genome using TopHat. A heatmap with highly variable genes across the samples was plotted using pheatmap package (pheatmap). The rlog transformed counts of each gene were centered across the samples. Ribosomal RNA genes and predicted genes that were increased in one sample ({"type":"entrez-nucleotide","attrs":{"text":"GC027190","term_id":"196521090","term_text":"GC027190"}}GC027190) were excluded from the rlog data used for heatmap since this increase was a result of an imperfect poly-A selection during the library preparation of this sample. Differential expression analysis was performed with DESeq2 (36). Differences in gene expression with a FDR adjusted p value below 0.1 were considered significant. Gene ontology analysis of differentially expressed genes was performed with clusterProfiler package (37). Motif analysis of the proximal promoter region (400 bp upstream of the transcription start site till 100 bp downstream) was performed using Homer software (Homer motif analysis). RNA-seq data were submitted to ArrayExpress (accession number E-MTAB-5921 (ArrayExpress URL)).