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Efficient low-cost chromatin profiling with CUT&Tag

Abstract

We recently introduced Cleavage Under Targets & Tagmentation (CUT&Tag), an epigenomic profiling strategy in which antibodies are bound to chromatin proteins in situ in permeabilized nuclei. These antibodies are then used to tether the cut-and-paste transposase Tn5. Activation of the transposase simultaneously cleaves DNA and adds adapters (‘tagmentation’) for paired-end DNA sequencing. Here, we introduce a streamlined CUT&Tag protocol that suppresses DNA accessibility artefacts to ensure high-fidelity mapping of the antibody-targeted protein and improves the signal-to-noise ratio over current chromatin profiling methods. Streamlined CUT&Tag can be performed in a single PCR tube, from cells to amplified libraries, providing low-cost genome-wide chromatin maps. By simplifying library preparation CUT&Tag requires less than a day at the bench, from live cells to sequencing-ready barcoded libraries. As a result of low background levels, barcoded and pooled CUT&Tag libraries can be sequenced for as little as $25 per sample. This enables routine genome-wide profiling of chromatin proteins and modifications and requires no special skills or equipment.

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Fig. 1: Steps in antibody-targeted chromatin profiling strategies.
Fig. 2: CUT&Tag provides high signal-to-noise ratios and reproducibility for native and lightly cross-linked cells and nuclei.
Fig. 3: Comparison of scCUT&Tag to single-cell ChIP-seq.
Fig. 4: Similar results are obtained using DNA extraction and single-tube CUT&Tag options.
Fig. 5: Suppression of accessible DNA tagmentation.
Fig. 6: CoBATCH and ACT-seq peaks correspond to ATAC-seq peak summits genome-wide.

Data availability

Publicly available datasets analyzed in this work are available in Supplementary Note 1. All sequencing data generated in this study have been deposited in GEO under accession GSE145187.

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Acknowledgements

We thank Christine Codomo for pooling Illumina sequencing libraries and members of our laboratory and colleagues at the Fred Hutch for providing input. We are especially grateful to the many Protocols.io subscribers around the world who have tried CUT&Tag and provided helpful comments and feedback that have enriched this protocol. This work was supported by the Howard Hughes Medical Institute (H.S.K.-O. and S.H.), grants R01 HG010492 (S.H.) and R01 GM108699 (K.A.) from the National Institutes of Health and an HCA Seed Network grant from the Chan-Zuckerberg Initiative (S.H.).

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Authors

Contributions

H.S.K.-O. and S.H. developed the protocol with input from K.A and D.H.J. S.H. performed the experiments, and with J.G.H. analyzed the data. S.H. and K.A. wrote the manuscript with input from H.S.K.-O, D.H.J., and J.G.H.

Corresponding author

Correspondence to Steven Henikoff.

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Competing interests

H.S.K.-O. and S.H. have filed patent applications related to this work.

Additional information

Peer review information Nature Protocols thanks Sabrina Krueger, Julia Zeitlinger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Kaya-Okur, H. S. et al. Nat. Commun. 10, 1930 (2019): https://doi.org/10.1038/s41467-019-09982-5

Supplementary information

Supplementary Information

Supplementary Note 1 and Supplementary Fig. 1.

Reporting Summary.

Supplementary Table 1

Primer Sequences.

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Kaya-Okur, H.S., Janssens, D.H., Henikoff, J.G. et al. Efficient low-cost chromatin profiling with CUT&Tag. Nat Protoc 15, 3264–3283 (2020). https://doi.org/10.1038/s41596-020-0373-x

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