MicroRNA Profiling of Melanoma Cell Lines

MicroRNA profiling was performed across the LM-MEL panel of cell lines by small RNA sequencing at the Australian Genome Research Facility (AGRF) on the Illumina HiSeq platform using total RNA, including small RNAs, purified from cell line pellets using the Qiagen miRNEasy isolation kit, following the manufacturer’s recommendations (Qiagen, Chadstone, Victoria, Australia). Library preparation and 5’-barcode multiplexing were performed prior to sequencing; each sample was run in 3–4 sequencing lanes as required to achieve adequate sequencing depth. Initial read quality assessment and filtering were performed by AGRF. De-multiplexed raw read data and quality scores were provided in fastq format. All reads for each sample were concatenated using the UNIX command line and collapsed to single fasta format files using the FASTX-Toolkit (v. 0.0.13) command-line tool FASTQ Collapser. Collapsed reads were processed through the miRanalyzer webserver [93 (link)] to map reads to the genome using the hg18 build of the UCSC Homo sapiens genome, followed by mapping of miRs to miRBase [94 –99 (link)]. Raw and processed miR abundance data were deposited on the Gene Expression Omnibus (GEO; dataset GSE89438; Additional file 1). These data underwent normalisation and a log₂-transformation was performed (Fig. 6c).