Investigating physical chromatin associations across the Xenopus genome by chromatin immunoprecipitation.
Chromatin immunoprecipitation (ChIP) combined with genomic analysis techniques provide a global snapshot of protein-DNA interactions in the context of chromatin, yielding insights into which genomic loci might be regulated by the DNA-associated protein under investigation. This protocol describes how to perform ChIP on intact or dissected Xenopus embryos. The ChIP-isolated DNA fragments are suitable for high-throughput sequencing (ChIP-Seq) or for quantitative PCR (ChIP-qPCR). In this protocol, embryonic tissue is harvested from Xenopus tropicalis or Xenopus laevis at the developmental stage of interest, and DNA-associated proteins are immobilized to their endogenous genomic binding sites with formaldehyde. Nuclei are extracted from embryos and subjected to sonication so as to shear the chromatin to a size that allows sufficient positional resolution of protein binding to genomic DNA. Chromatin fragments bound by the protein of interest are immunoprecipitated using antibody-coupled beads, washed under high-stringency conditions, and stripped from the beads with anionic detergents. The chemical cross-links are reversed, and the coimmunoprecipitated DNA is purified. The resulting DNA fragments can be analyzed by qPCR or used to create a ChIP-Seq library. General advice for qPCR and for making ChIP-Seq libraries is offered, and approaches for analyzing ChIP-Seq data are outlined.
Division of Systems Biology, National Institute for Medical Research, London NW7 1AA, United Kingdom.
George E
GE
Gentsch
James C
JC
Smith
D000818:Animals; D002843:Chromatin / Q000302:isolation & purification*; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D005786:Gene Expression Regulation; D056426:Genetic Loci; D016678:Genome; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D060888:Real-Time Polymerase Chain Reaction; D013010:Sonication; D016707:Tissue Fixation; D014981:Xenopus / Q000196:embryology*
D002843:Chromatin