ChIP-on-chip Protocol for Genome-Wide Analysis of Transcription Factor Binding in Drosophila Melanogaster Embryos

Nat Protoc. 2006;1(6):2839-55. doi: 10.1038/nprot.2006.383.

Abstract

This protocol describes a method to detect in vivo associations between proteins and DNA in developing Drosophila embryos. It combines formaldehyde crosslinking and immunoprecipitation of protein-bound sequences with genome-wide analysis using microarrays. After crosslinking, nuclei are enriched using differential centrifugation and the chromatin is sheared by sonication. Antibodies specifically recognizing wild-type protein or, alternatively, a genetically encoded epitope tag are used to enrich for specifically bound DNA sequences. After purification and polymerase chain reaction-based amplification, the samples are fluorescently labeled and hybridized to genomic tiling microarrays. This protocol has been successfully used to study different tissue-specific transcription factors, and is generally applicable to in vivo analysis of any DNA-binding proteins in Drosophila embryos. The full protocol, including the collection of embryos and the collection of raw microarray data, can be completed within 10 days.

MeSH terms

  • Animals
  • Binding Sites
  • Data Interpretation, Statistical
  • Drosophila melanogaster / embryology
  • Drosophila melanogaster / genetics
  • Drosophila melanogaster / metabolism*
  • Embryo, Nonmammalian
  • Genome, Insect*
  • Genomics / methods*
  • Microarray Analysis / methods*
  • Polymerase Chain Reaction
  • Quality Control
  • Research Design
  • Transcription Factors / metabolism*

Substances

  • Transcription Factors