Computational protocol: Zeb2 Recruits HDAC-NuRD to Inhibit Notch and Controls Schwann Cell Differentiation and Remyelination

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Protocol publication

[…] RNA from P7 control and Zeb2 cKO sciatic nerves were extracted using TRIZOL (Life Technologies) followed by purification using an RNeasy Mini Kit (Qiagen). RNA-seq libraries were prepared using Illumina RNA-Seq Preparation Kit and sequenced by HiSeq 2000 sequencer. RNA-seq reads were mapped using TopHat with default settings (http://tophat.cbcb.umd.edu). TopHat output data were then analyzed by Cufflinks to (1) calculate FPKM values for known transcripts in mouse genome reference, and (2) test the changes of gene expression between Zeb2 cKO and control. Gene Ontology (GO) analysis was performed using The Database for Annotation, Visualization and Integrated Discovery (DAVID; http://david.abcc.ncifcrf.gov). The heatmap was generated based on log2 [FPKM] by AltAnalyze , with normalization of rows relative to row mean. [...] RNA-seq libraries were prepared using Illumina RNA-Seq Preparation Kit and sequenced by a HiSeq 2000 sequencer. All RNA-Seq data were aligned to mm9 using TopHat with default settings. We used Cuff-diff to (1) estimate FPKM values for known transcripts and (2) analyze differentially expressed transcripts. In all differential expression tests, a difference was considered significant if the q value was less than 0.05 (Cuff-diff default). Heatmap of gene expression was generated using R language (http://www.r-project.org). GO-analysis of gene expression changes was performed using Gene Set Enrichment (GSEA, http://www.broadinstitute.org/gsea/index.jsp). We used ToppCluster (https://toppcluster.cchmc.org/) to construct the network of genes belonging to over-represented GO-term categories. […]

Pipeline specifications

Software tools TopHat, Cufflinks, DAVID, AltAnalyze, GSEA, ToppCluster
Application RNA-seq analysis
Organisms Mus musculus
Diseases Peripheral Nervous System Diseases
Chemicals Zinc