Computational protocol: How asbestos drives the tissue towards tumors: YAP activation, macrophage and mesothelial precursor recruitment, RNA editing, and somatic mutations

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Protocol publication

[…] Read data were quality controlled with Fastqc and FastqScreen to confirm that reads are of high quality and free of contaminants. Read-alignment was done using the STAR-aligner []. As reference we used the Ensembl genome build GRCm38.p3. Gene expression values were computed with the function featureCounts from the R package Rsubread []. Differential expression was computed using the generalized linear model implemented in the Bioconductor package DESeq2 [].Variant analysis was performed using the GATK (software.broadinstitute.org) software following the RNA-seq best practices. Specifically, we looked at variants on transcript regions and ignored sites with <2 reads supporting the SNV. We also excluded variants in Ig loci since they are generated by well known somatic hypermutation mechanisms which would be a potentially confounding effect. In order to identify A to G mutation due to RNA-editing events, predicted A to I sites from http://rnaedit.com/download/ were translated to mm10 coordinates. Gene set expression enrichment analysis was performed using online available software (http://software.broadinstitute.org/gsea/msigdb/index.jsp) [].Expression changes in mouse tumors compared to non-tumor tissues were compared to expression changes in the study GSE51024 [] which compared mesothelioma tumors vs. normal tissue. The analysis was performed with Geo2R, which makes use of the R/Bioconductor package limma. Log2 fold changes, nominal p values, and Benjamini–Hochberg FDR were calculated by Geo2R/Limma.FPKM data corresponding to gene symbols within given GO terms were retrieved from the normalized data frame, if the p value was <0.01 and the absolute value of the log2ratio was >1 for the comparison among tum and asbestos-exposed tissue and mean FPKM was greater than 1 in at least one of the three sample groups.Selected gene expression analysis (primers in Supplementary Table ) was performed as previously described []. […]

Pipeline specifications

Software tools FastQC, STAR, Subread, DESeq2, GATK, GSEA, GEO2R, limma
Databases MSigDB
Applications RNA-seq analysis, Transcription analysis
Organisms Mus musculus
Diseases Neoplasms
Chemicals Asbestos