RNAseq experiment

Total RNA from melanoma cells was isolated using the total RNA Kit (Machery & Nagel) and used for paired-end RNA-seq. Quality was assessed with an Agilent 2100 Bioanalyzer (RIN > 9.5). For library preparation, polyA capture from 100 ng of total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB) was done. Libraries were prepared using the Ultra II Directional RNA Library Prep Kit for Illumina according to the manufacturer’s instructions. The libraries were denatured, diluted to 270 pM and sequenced as paired-end 100 bp reads on an Illumina NovaSeq 6000 platform at a depth of approximately 25 mio reads each. The read quality of RNA-seq data in fastq files was assessed using FastQC (v0.11.4). Reads were aligned using STAR (v2.7.0a), allowing gapped alignments to account for splicing against the Ensembl H. sapiens genome v95. Alignment quality was analyzed using samtools (v1.1). Normalized read counts for all genes were obtained using BEAVR [52]. Transcripts covered with more than 10 reads in at least one group were analyzed to determine differential expression. We set |log2 fold-change| ≥ 0.5 and FDR ≤ 0.05 to call differentially expressed genes. Gene-level abundances were derived as normalized count per million and used for calculating the log2-transformed expression changes underlying the expression heatmaps for which ratios were computed against mean expression in control samples.