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Using ChIP-Based Approaches to Characterize FOXO Recruitment to its Target Promoters

  • Neeraj Kumar
  • Arnab MukhopadhyayEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1890)

Abstract

Chromatin immunoprecipitation (ChIP) coupled to quantitative real-time PCR (ChIP-qPCR) or Next-Generation Sequencing (ChIP-seq) enables us to study the dynamics of chromatin recruitment of transcription factors (TFs). The popular model system Caenorhabditis elegans has provided us with fundamental understanding of the role of Insulin/IGF-1-like signaling (IIS) in metabolism and aging. The FOXO TF DAF-16 is the major output of the pathway that regulates most of the phenotypes associated with the IIS pathway. Here, we describe a ChIP protocol to study FOXO recruitment dynamics in whole C. elegans extracts. We discuss detailed practical procedures, including optimization, growth, harvesting, formaldehyde fixation, sonication of worms, TF immunoprecipitation for further downstream processing using qPCR as well as NGS for the analysis of FOXO-bound DNA.

Key words

Chromatin immunoprecipitation Quantitative real-time PCR Next-generation sequencing Transcription factor FOXO DAF-16 Gene promotor C. elegans 

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Reproductive BiologyAll India Institute of Medical Sciences (AIIMS)New DelhiIndia
  2. 2.Molecular Aging LaboratoryNational Institute of ImmunologyNew DelhiIndia

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