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Identifying Direct Downstream Targets: WT1 ChIP-Seq Analysis

  • Fabio da Silva
  • Filippo Massa
  • Andreas SchedlEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1467)

Abstract

Identifying targets of transcriptional regulators such as the Wilms’ tumor-suppressor protein (WT1) is an integral part of understanding the mechanisms governing the spatial and temporal activation of different genes. A commonly used strategy for studying transcription factors involves performing chromatin immunoprecipitation (ChIP) for the protein of interest with an appropriate antibody in crosslinked cells. Following ChIP, the enriched DNA is sequenced using next-generation sequencing (NGS) technologies and the transcription factor target sites are identified via bioinformatics analysis. Here we provide a detailed protocol for performing a successful ChIP-Seq experiment for WT1. We have optimized and simplified the several steps necessary for the immunoprecipitation of WT1’s target-binding sites. We also suggest several strategies for validating the experiment and provide brief guidelines on how to analyze the large amounts of data generated from high-throughout sequencing. This method can be adapted for a variety of different tissues and/or cell types to help understand the role of WT1 in regulating gene expression.

Key words

Wilms’ tumor suppressor 1 (WT1) Next-generation sequencing (NGS) Chromatin immunoprecipitation (ChIP) 

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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Fabio da Silva
    • 1
    • 2
    • 3
  • Filippo Massa
    • 1
    • 2
    • 3
  • Andreas Schedl
    • 1
    • 2
    • 3
    Email author
  1. 1.Institute of Biology ValroseUniversité de Nice-SophiaNiceFrance
  2. 2.Inserm, UMR1091NiceFrance
  3. 3.CNRS, UMR7277NiceFrance

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