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Pipeline publication

[…] romega). The size range of final cDNA libraries was determined applying the DNA 1000 chip on the Bioanalyzer 2100, (Agilent; 290-310 bp). cDNA libraries were amplified and sequenced via cBot and HiSeq 2000 (Illumina; SR, 1×50 bp, 6 Gb/sample ca. 30 million reads per sample). Sequence images were transformed with Illumina software BaseCaller to bcl files, which were demultiplexed to fastq files with CASAVA (version 1.8.2). Quality check was performed via FastQC (version 0.10.1, Babraham Bioinformatics). Fastq files were mapped to the human reference transcriptome (UCSC hg19) using Tophat (Galaxy Version 0.9) [] Read counts for each sample and each gene were aggregated using a htseq-count []. DESeq2 (version 1.10.1) was used for measuring differential expression []. RNA library preparation and sequencing was done by the Transcriptome Analysis Laboratory (TAL, Göttingen)., Gene set enrichment analysis (GSEA) from C2 curated gene sets (provided by the Molecular Signatures Database (MSigDB) v5.0) was performed using variance stabilized normalized read counts. [, ]. The threshold of significant enrichment (q≤0.25) was implied according to the GSEA standards (http://www.broadinstitute.org/gsea/doc/GSEAUserGuideFrame.html)., Raw data for p53 ChIP-Sequencing [] were downloaded from the Gene Omibus database (ID GSE47043). The reads were mapped to the human reference genome (UCSC hg19) using Bowtie (version 1.0.0) []. Peak calling was done by Model-based Analysis of ChIP-Seq (version 1.4.2 []. Coverage was determined by normalizing the total number of mapped reads per hundred million. p53 enrichment was analyzed on the transcriptional start sites (TSSs) of genes that were upregulated in MCF-7 cells after Nutlin and after Nutlin+Wip1i treatment using deeptools functions [] based on the Galaxy framework []., Cells were seeded in 6-well plates and treated with drugs, At 24h post-treatment, cells were harvested (inclusive of medium) and centrifuged at 1500xg for 5 min at 4°C. The pelleted cells were resuspended in 250μl caspase lysis buffer (1M Tris-HCl, 2mM MgCl2, 150mM NaCl, 10mM DTT, protease-inhibitor (Roche complete mini)). They were shock-frozen thrice in liquid nitrogen and centrifuged at 15,000xg for 15 min at 4°C. 40μl of lysate was pipetted per well in a 96-well plate in triplicates. 10μl of Ac-DEVD-AMC substrate (working concentration 25μM) (ALX-260-031 Enzo) was added to each sample. Caspase activity was measured using a fluorom […]

Pipeline specifications

Software tools DESeq2, GSEA, Bowtie, deepTools