Native internally calibrated chromatin immunoprecipitation for quantitative studies of histone post-translational modifications


Chromatin 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.

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Fig. 1: The ICeChIP–seq workflow.
Fig. 2: ICeChIP–seq bioinformatics analysis pipeline.
Fig. 3: Representative results of the ICeChIP procedure.

Data accessibility

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. The most updated versions of the tools and scripts described in this work can be found at


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This work was funded by the National Institutes of Health under award number R01-GM115945 to A.J.R.

Author information

A.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–seq analyses to validate this protocol. R.N.S. wrote the manuscript with input from the other authors.

Correspondence to Alexander J. Ruthenburg.

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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 information

Peer 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’s 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, 886–899 (2015):

Werner, M. S. et al. Nat. Struct. Mol. Biol. 24, 596–603 (2017):

Shah, R. N. et al. Mol. Cell 72, 162–177 (2018):

Supplementary information

Supplementary Table 1

Primer and probe sequences

Reporting Summary

Supplementary Table 2

Example of calibration table formatting

Supplementary Table 3

Hamming distances for NEBNext Illumina adaptors

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Grzybowski, A.T., Shah, R.N., Richter, W.F. et al. Native internally calibrated chromatin immunoprecipitation for quantitative studies of histone post-translational modifications. Nat Protoc 14, 3275–3302 (2019).

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