Computational protocol: Cancer Drug Addiction is Relayed by an ERK2-Dependent PhenotypeSwitch

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Protocol publication

[…] RNA was isolated using Trizol, and cDNA libraries were sequenced on an Illumina HiSeq2000 to obtain 65 bp single-end sequence reads. Reads were aligned to the GRCh38 human reference genome using Tophat (2.1.0), and gene counts were obtained using HTSeq-count. All downstream analyses were performed in R using the Bioconductor framework. DESeq2 rlog-values were used for visualization of gene expression levels in heat maps. Heat maps represent mean rlog-values of two independent biological replicate experiments. Clustering was performed using default settings of the heatmap.2 function in the gplots package. For , gene-wise scaling was applied to rlog-values, and the 200 most variable are represented. For and , rlog-values were mean normalized on a gene-wise basis. For gene set enrichment analyses, counts per million (cpm) were used as input. For and , time points were converted to a ranking score (0, 1, 2 or 3 for time points 0, 6, 24 or 48 h of A375BMR and 0, 1, 3 or 5 days of 451LuBR, respectively); for , the extent of rescue from cell death was converted to a ranking score (1, 2, 3, or 4 for Mel888BMR cells expressing sgCtrl, sgJUNB, or sgERK2 in the context of drug removal, and Mel888BMR cells with treatment, respectively). Pre-ranked gene lists were created by correlating these ranking scores with gene expression using the Spearman's rank correlation coefficient metric. Phenotype switching and MITF target gene lists were obtained from The TEAD and AP1 signatures were taken from Verfaillie et al., and the JUNB signature was derived from of Fontana et al., where human homologs of mouse genes were derived using the biomaRt package. Analyses were performed using javaGSEA, and replotted from the output of this tool using the replotGSEA function ( […]

Pipeline specifications

Software tools TopHat, HTSeq, DESeq2, gplots
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus
Diseases Lung Neoplasms, Melanoma, Neoplasms, Lymphoma, B-Cell, Substance-Related Disorders
Chemicals Dacarbazine, Tyrosine