A375 cells were treated for 24 hours with 100nM trametinib or DMSO. RNA-seq libraries were prepared from total RNA using the TruSeq RNA Sample Prep Kit (Illumina, Inc., San Diego, CA, USA) and a Sciclone NGSx Workstation (PerkinElmer, Waltham, MA, USA). Library size distributions were validated using an Agilent 2200 TapeStation (Agilent Technologies). Sequencing was performed using an Illumina HiSeq 2500 employing a paired-end, 50 base read length (PE50) approach. Image analysis and base calling were performed using Illumina's Real Time Analysis v1.18 software, followed by ‘demultiplexing’ of indexed reads and generation of FASTQ files, using Illumina's bcl2fastq Conversion Software v1.8.4 (http://support.illumina.com/downloads/bcl2fastq_conversion_software_184.html). Low quality reads were filtered prior to alignment to the reference genome (UCSC hg38 assembly) using TopHat v2.1.028 (link). Counts were generated from TopHat alignments for each gene using the Python package HTSeq v0.6.129 (link). Genes which did not have at least 1 CPM in at least 1 sample were discarded, followed by TMM normalization using the Bioconductor package edgeR v3.12.0.30 (link). LogFC and logCPM values were calculated from the normalized results.