AbstractChromatin immunoprecipitation coupled to next-generation sequencing (ChIP-seq) has served as the central method for the study of histone modifications for the past decade. In ChIP-seq analyses, antibodies selectively capture nucleosomes bearing a modification of interest and the associated DNA is then mapped to the genome to determine the distribution of the mark. This approach has several important drawbacks: (i) ChIP interpretation necessitates the assumption of perfect antibody specificity, despite growing evidence that this is often not the case. (ii) Common methods for evaluating antibody specificity in other formats have little or no bearing on specificity within a ChIP experiment. (iii) Uncalibrated ChIP is reported as relative enrichment, which is biologically meaningless outside the experimental reference frame defined by a discrete immunoprecipitation (IP), thus preventing facile comparison across experimental conditions or modifications. (iv) Differential library amplification and loading onto next-generation sequencers, as well as computational normalization, can further compromise quantitative relationships that may exist between samples. Consequently, the researcher is presented with a series of potential pitfalls and is blind to nearly all of them. Here we provide a detailed protocol for internally calibrated ChIP (ICeChIP), a method we recently developed to resolve these problems by spike-in of defined nucleosomal standards within a ChIP procedure. This protocol is optimized for specificity and quantitative power, allowing for measurement of antibody specificity and absolute measurement of histone modification density (HMD) at genomic loci on a biologically meaningful scale enabling unambiguous comparisons. We provide guidance on optimal conditions for next-generation sequencing (NGS) and instructions for data analysis. This protocol takes between 17 and 18 h, excluding time for sequencing or bioinformatic analysis. The ICeChIP procedure enables accurate measurement of histone post-translational modifications (PTMs) genome-wide in mammalian cells as well as Drosophila melanogaster and Caenorhabditis elegans, indicating suitability for use in eukaryotic cells more broadly. Subscription info for Chinese customersWe have a dedicated website for our Chinese customers. Please go to naturechina.com to subscribe to this journal.Go to naturechina.comRent or Buy articleGet time limited or full article access on ReadCube.from$8.99Rent or BuyAll prices are NET prices. The sequencing data described in this work have previously been published 43,45. Sequencing data can be found at GEO under accession numbers GSE60378 and GSE103543. Code availability All code used in this work is listed under Analytical tools. Scripts provided here are under GNU General Public License. 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ShahDepartment of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USAAlexander J. RuthenburgAuthorsAdrian T. GrzybowskiView author publicationsYou can also search for this author in PubMed聽Google ScholarRohan N. ShahView author publicationsYou can also search for this author in PubMed聽Google ScholarWilliam F. RichterView author publicationsYou can also search for this author in PubMed聽Google ScholarAlexander J. RuthenburgView author publicationsYou can also search for this author in PubMed聽Google ScholarContributionsA.T.G. and A.J.R. conceived and developed ICeChIP. A.T.G. and R.N.S. wrote computational scripts for data analysis while A.J.R. provided oversight. W.F.R. independently conducted ICeChIP鈥搒eq analyses to validate this protocol. R.N.S. wrote the manuscript with input from the other authors.Corresponding authorCorrespondence to Alexander J. Ruthenburg.Ethics declarations Competing interests A.J.R. and A.T.G. hold partial intellectual property rights to ICeChIP as inventors. R.N.S., A.T.G. and A.J.R. have previously served in a compensated consulting role to Epicypher, the commercial developer and supplier of ICeChIP barcoded nucleosomes (SNAP-ChIP and CAP-ChIP, both of which are under a license of the University of Chicago (patent #US20160341743)). Additional informationPeer review information Nature Protocols thanks Jeff Dilworth, Alon Goren, Yuting Liu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.Publisher鈥檚 note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Related links Key references using this protocol Grzybowski, A. T., Chen, Z. Ruthenburg, A. J. Mol. Cell 58, 886鈥?99 (2015): https://doi.org/10.1016/j.molcel.2015.04.022Werner, M. S. et al. Nat. Struct. Mol. Biol. 24, 596鈥?03 (2017): https://doi.org/10.1038/nsmb.3424Shah, R. N. et al. Mol. Cell 72, 162鈥?77 (2018): https://doi.org/10.1016/j.molcel.2018.08.015Supplementary information CommentsBy submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Sign up for the Nature Briefing newsletter 鈥?what matters in science, free to your inbox daily.