Genome-wide snapshot of chromatin regulators and states in Xenopus embryos by ChIP-Seq.
The recruitment of chromatin regulators and the assignment of chromatin states to specific genomic loci are pivotal to cell fate decisions and tissue and organ formation during development. Determining the locations and levels of such chromatin features in vivo will provide valuable information about the spatio-temporal regulation of genomic elements, and will support aspirations to mimic embryonic tissue development in vitro. The most commonly used method for genome-wide and high-resolution profiling is chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq). This protocol outlines how yolk-rich embryos such as those of the frog Xenopus can be processed for ChIP-Seq experiments, and it offers simple command lines for post-sequencing analysis. Because of the high efficiency with which the protocol extracts nuclei from formaldehyde-fixed tissue, the method allows easy upscaling to obtain enough ChIP material for genome-wide profiling. Our protocol has been used successfully to map various DNA-binding proteins such as transcription factors, signaling mediators, components of the transcription machinery, chromatin modifiers and post-translational histone modifications, and for this to be done at various stages of embryogenesis. Lastly, this protocol should be widely applicable to other model and non-model organisms as more and more genome assemblies become available.
Division of Systems Biology, MRC National Institute for Medical Research; ggentsc@nimr.mrc.ac.uk.
George E
GE
Gentsch
Division of Systems Biology, MRC National Institute for Medical Research.
Ilya
I
Patrushev
Division of Systems Biology, MRC National Institute for Medical Research.
James C
JC
Smith
D000818:Animals; D002843:Chromatin / Q000235:genetics* / Q000378:metabolism; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000032:analysis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D055106:Genome-Wide Association Study / Q000379:methods; D023281:Genomics / Q000379:methods; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006657:Histones / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D011499:Protein Processing, Post-Translational; D014157:Transcription Factors / Q000235:genetics; D014981:Xenopus / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism
D002843:Chromatin; D004268:DNA-Binding Proteins; D006657:Histones; D014157:Transcription Factors; D004247:DNA