Computational protocol: Zika virus has oncolytic activity against glioblastoma stem cells

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Protocol publication

[…] 5-µm-thick sections of paraffin-embedded tissues were analyzed for hematoxylin and eosin (H&E; Thermo Fisher), Picro-Sirius Red (Sigma-Aldrich), and Masson’s Trichrome (Diagnostic Biosystems) according to the manufacturer’s instructions. 4×, 10×, and 20× images were captured on an Eclipse 80i brightfield microscope (Nikon). Image analysis was performed by thresholding for positive staining and normalizing to total tissue area using ImageJ (NIH) and MetaMorph v7.7.0.0 (Molecular Devices) software (). [...] RNA was obtained from GSCs infected with ZIKV-Dakar for 36–48 h. Total cellular RNA was isolated using the RNeasy kit (Qiagen). RNA-seq reads were aligned to the Ensembl release 76 assembly with STAR v. 2.5.1a. Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:feature Count v. 1.4.6p5. Transcript counts were produced by Sailfish v. 0.6.3. Sequencing performance was assessed for total number of aligned reads, total number of uniquely aligned reads, genes and transcripts detected, ribosomal fraction, known junction saturation, and read distribution over known gene models with RSeQC v. 2.6.2.All gene-level and transcript counts were imported into the R/Bioconductor package EdgeR, and TMM normalization size factors were calculated to adjust for samples for differences in library size. Genes or transcripts not expressed in any sample were excluded from further analysis. The TMM size factors and the matrix of counts were imported into R/Bioconductor package Limma, and weighted likelihoods based on the observed mean–variance relationship of every gene/transcript were calculated for all samples with the Voom function. Performance of the samples was assessed with a Spearman correlation matrix and multidimensional scaling plots. Gene/transcript performance was assessed with plots of residual standard deviation of every gene to their mean log count with a robustly fitted trend line of the residuals. Generalized linear models were created to test for gene- or transcript-level differential expression. Differentially expressed genes and transcripts were then filtered for FDR-adjusted P values less than or equal to 0.05.To enhance the biological interpretation of the large set of transcripts, grouping of genes/transcripts based on functional similarity was achieved using the R/Bioconductor packages GAGE and Pathview. GAGE and Pathview were also used to generate pathway maps on known signaling and metabolism pathways curated by KEGG.For the matched GSC and DGC lines, RNA-seq data were evaluated for type I and II IFN signatures between GSCs and DGCs using gene set enrichment analysis and were validated using RNA-seq data on additional matched cell lines derived from . IFN signatures were derived from the Molecular Signature Database curated by the Broad Institute (; http://www.broad.mit.edu/gsea/). Unsupervised hierarchical clustering was performed using FPKM values from matched TPC and DGC lines. All RNA-seq data are available through the Gene Expression Omnibus (GEO) data repository under accession nos. GSE102244 and GSE102924. […]

Pipeline specifications

Software tools ImageJ, MetaMorph
Application Microscopic phenotype analysis
Organisms Zika virus, Human poliovirus 1 Mahoney, Homo sapiens, Mus musculus
Diseases Brain Neoplasms, Glioblastoma, Neoplasms, HIV Infections