Identifying direct downstream targets: WT1 chIP-Seq analysis

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 identifi ed via bioinformatics analysis. Here we provide a detailed protocol for performing a successful ChIP-Seq experiment for WT1. We have optimized and simplifi ed 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.