Computational protocol: Autophagy protects ovarian cancer-associated fibroblasts against oxidative stress

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Protocol publication

[…] Double stranded cDNA were purified and sonicated with Diagenode Bioruptor to obtain DNA fragments of 200 to 400 bp. The fragmented DNA was then A-tailed after end repair, ligated to the adaptors and PCR amplified according to the instructions of illumina Nextera DNA Sample Prep Kit. The NGS libraries were sequenced on the illumina GA IIx (single end, 75 bp length). The sequencing depth was 29 M and 23 M reads for NFs and CAFs, respectively. After mapping to the human genome with TopHat software, the FPKM (fragments per kilobase of transcript per million fragments mapped) of NFs versus CAFs and their Log2 fold change was calculated with Cufflinks software. Then the Log2 fold change of NFs vs. CAFs was used to generate the pre-ranked gene list and loaded to do the GSEA (gene set enrichment anylysis) with the gene set database c5.all.v3.0.symbols.gmt downloaded from MSigDB. […]

Pipeline specifications

Software tools TopHat, Cufflinks, GSEA
Databases MSigDB
Application RNA-seq analysis
Diseases Ovarian Neoplasms
Chemicals Cisplatin, Hydrogen Peroxide, Oxygen, Superoxides, Lactic Acid