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Measurement of differential chromatin interactions with absolute quantification of architecture (AQuA-HiChIP)


Methods developed to capture protein-anchored chromatin interactions (chromatin interaction analysis by paired-end tag sequencing and HiChIP) have yielded tremendous insights into the 3D folding principles of the genome, but are normalized by sequencing depth and therefore unable to accurately measure global changes in chromatin interactions and contact domain organization. We herein describe the protocol for absolute quantification of chromatin architecture (AQuA)–HiChIP, an advance that allows the absolute differences in protein-anchored chromatin interactions between samples to be determined. With our method, defined ratios of mouse and human fixed nuclei are mixed and subjected to endonuclease digestion. Chromatin contacts are captured by biotin-dATP incorporation and proximity ligation, followed by gentle shearing, ChIP, biotin capture and paired-end sequencing. 3D contacts are counted from paired-end tags (PETs) from the human genome and are normalized to the total PETs from the mouse genome. As orthogonal normalization allows observation of global changes, the approach will enable more quantitative insights into the topological determinants of transcriptional control and tissue-specific epigenetic memory. With our approach, we have discovered that rapid histone deacetylase inhibition disrupts super enhancer function by creating many new aberrant contacts. The code for data analysis is available at This protocol reports both experimental and bioinformatic details to perform AQuA-HiChIP, going from cell culture to ranking chromatin interactions within 6 d.

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Fig. 1: AQuA-HiChIP experimental schematic overview.
Fig. 2: Comparison of HiChIP and AQuA-HiChIP between treated and untreated cells reveals the difference between apparent and absolute contact changes.
Fig. 3: AQuA-HiChIP size distribution.
Fig. 4: Bioinformatic analysis example plots.
Fig. 5: AQuA-HiChIP contact frequency calculations.
Fig. 6: AQuA-HiChIP reveals target-dependent architectural changes.
Fig. 7: Global AQuA normalization does not influence H3K27ac HiChIP contact frequencies at H3K27me3 domains.

Data availability

Data generated by this protocol, and visualized herein, is available through Gene Expression Omnibus, accession number GSE120770.

Code availability

All code used herein is either provided by other research laboratories (see links throughout the protocol for any given step) or is custom scripted in R (available here: The code in this protocol has been peer reviewed.


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We gratefully acknowledge Y. Song and the NCI Sequencing Core for assistance. We are grateful to Tom Misteli and Justin Demmerle for helpful conversations relating to this work. We thank Emma Chory for technical advice. We wish to honor the lasting memory of Joseph P. Calarco. This work was facilitated by Biowulf High Performance Computing Systems and enabled by funding from the Division of Intramural Research from NIH NCI CCR.

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Authors and Affiliations



B.Z.S, B.E.G. and J.K. conceived of the project. B.Z.S. and B.E.G. performed AQuA-HiChIP experiments. B.E.G. built the AQuA analysis pipeline, B.Z.S. and B.E.G. wrote the paper.

Corresponding authors

Correspondence to Berkley E. Gryder, Javed Khan or Benjamin Z. Stanton.

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The authors declare no competing interests.

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Gryder, B. E. et al. Nat. Genet. 51, 1714–1722 (2019):

Stanton, B. et al. Preprint at (2018):

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Gryder, B.E., Khan, J. & Stanton, B.Z. Measurement of differential chromatin interactions with absolute quantification of architecture (AQuA-HiChIP). Nat Protoc 15, 1209–1236 (2020).

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