RNA was isolated using an All-Prep DNA/RNA Kit (Qiagen) following the manufacturer's instructions. RNA samples were sent to the Deep Sequencing Core Facility in Göttingen, where samples were prepared using TrueSeq Kits (Illumina) and HiSeq_4000 performing 50 million reads/sample.
Fastq-files were quality checked with FastQC (Babraham Bioinformatics) and trimmed for residual adapter sequences. Reads were aligned to the GRCh38 human genome using TopHatR (2.1.0–Johns Hopkins University, Center for Computational Biology) and Bowtie2 (46 (link)). Counts per gene were determined as sum of all reads mapped within a gene region. Principal component (PC) analysis was performed in R (47 ) using the 1,000 top-variable genes within the data set. Differentially expressed genes were identified using negative binomial distributions as implemented in the DESeq2 package (48 (link)) in R. False discovery rates (FDR) were calculated to adjust p-values for multiple testing and FDR-values below 0.05 were considered as significant. Expression data for differentially expressed genes were variance-stabilized transformed and scaled prior to visualization in heat maps. RNA sequencing data are available at the GEO platform with the accession number GSE129196.
Fastq-files were quality checked with FastQC (Babraham Bioinformatics) and trimmed for residual adapter sequences. Reads were aligned to the GRCh38 human genome using TopHatR (2.1.0–Johns Hopkins University, Center for Computational Biology) and Bowtie2 (46 (link)). Counts per gene were determined as sum of all reads mapped within a gene region. Principal component (PC) analysis was performed in R (47 ) using the 1,000 top-variable genes within the data set. Differentially expressed genes were identified using negative binomial distributions as implemented in the DESeq2 package (48 (link)) in R. False discovery rates (FDR) were calculated to adjust p-values for multiple testing and FDR-values below 0.05 were considered as significant. Expression data for differentially expressed genes were variance-stabilized transformed and scaled prior to visualization in heat maps. RNA sequencing data are available at the GEO platform with the accession number GSE129196.