title: Calibrating ChIP-Seq with Nucleosomal Internal Standards to Measure Histone Modification Density Genome Wide.
abstract: Chromatin immunoprecipitation (ChIP) serves as a central experimental technique in epigenetics research, yet there are serious drawbacks: it is a relative measurement, which untethered to any external scale obscures fair comparison among experiments; it employs antibody reagents that have differing affinities and specificities for target epitopes that vary in abundance; and it is frequently not reproducible. To address these problems, we developed Internal Standard Calibrated ChIP (ICeChIP), wherein a native chromatin sample is spiked with nucleosomes reconstituted from recombinant and semisynthetic histones on barcoded DNA prior to immunoprecipitation. ICeChIP measures local histone modification densities on a biologically meaningful scale, enabling unbiased trans-experimental comparisons, and reveals unique insight into the nature of bivalent domains. This technology provides in situ assessment of the immunoprecipitation step, accommodating for many experimental pitfalls as well as providing a critical examination of untested assumptions inherent to conventional ChIP.
authors.affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
authors.forename: Adrian T
authors.initials: AT
authors.lastname: Grzybowski
authors.affiliation: Department of Chemistry, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.
authors.forename: Zhonglei
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Department of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA; Department of Biochemistry and Molecular Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA. Electronic address: aruthenburg@uchicago.edu.
authors.forename: Alexander J
authors.initials: AJ
authors.lastname: Ruthenburg
mesh_terms: D000818:Animals; D002138:Calibration; D002460:Cell Line; D047369:Chromatin Immunoprecipitation / Q000592:standards*; D004331:Drosophila melanogaster; D016678:Genome; D057809:HEK293 Cells; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008745:Methylation; D051379:Mice; D009707:Nucleosomes / Q000235:genetics*; D011499:Protein Processing, Post-Translational; D012015:Reference Standards; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
chemical_list: D006657:Histones; D009707:Nucleosomes