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Transcription factor chromatin profiling genome-wide using uliCUT&RUN in single cells and individual blastocysts


Determining chromatin-associated protein localization across the genome has provided insight into the functions of DNA-binding proteins and their connections to disease. However, established protocols requiring large quantities of cell or tissue samples currently limit applications for clinical and biomedical research in this field. Furthermore, most technologies have been optimized to assess abundant histone protein localization, prohibiting the investigation of nonhistone protein localization in low cell numbers. We recently described a protocol to profile chromatin-associated protein localization in as low as one cell: ultra-low-input cleavage under targets and release using nuclease (uliCUT&RUN). Optimized from chromatin immunocleavage and CUT&RUN, uliCUT&RUN is a tethered enzyme-based protocol that utilizes a combination of recombinant protein, antibody recognition and stringent purification to selectively target proteins of interest and isolate the associated DNA. Performed in native conditions, uliCUT&RUN profiles protein localization to chromatin with low input and high precision. Compared with other profiling technologies, uliCUT&RUN can determine nonhistone protein chromatin occupancies in low cell numbers, permitting the investigation into the molecular functions of a range of DNA-binding proteins within rare samples. From sample preparation to sequencing library submission, the uliCUT&RUN protocol takes <2 d to perform, with the accompanying data analysis timeline dependent on experience level.

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Fig. 1: Overview of sample preparation for uliCUT&RUN. uliCUT&RUN is amenable to many sources of low-input material.
Fig. 2: Overview of the uliCUT&RUN approach.
Fig. 3: Overview of the uliCUT&RUN bioinformatic analysis pipeline.
Fig. 4: Example quality controls for uliCUT&RUN libraries.
Fig. 5: Expected results for 50 ES cell uliCUT&RUN for TFs.
Fig. 6: Expected results for CTCF blastocyst or single-cell uliCUT&RUN.

Data availability

The GEO accession number for the sequencing data reported in this paper is GSE111121, originally generated for ref. 13.

Code availability

Code for sequencing data analysis is available at


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We thank the Henikoff group for the original development of CUT&RUN and discussion regarding application. We thank A. Boskovic and T. Fazzio for assistance in development and application of uliCUT&RUN. We thank members of the Hainer lab for helpful comments on the manuscript. This work was supported by the National Institutes of Health grant R35GM133732 to S.J.H.

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S.J.H. optimized the protocol and performed experiments. B.J.P. analyzed the data with assistance from S.J.H., B.J.P. and S.J.H wrote the manuscript.

Corresponding author

Correspondence to Sarah J. Hainer.

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The authors declare no competing interests.

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

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Key references using this protocol

Hainer, S. et al. Cell 177, 1319–1329.e11 (2019):

Xu, J. et al. Nat. Commun. 11, 1899 (2020):

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Patty, B.J., Hainer, S.J. Transcription factor chromatin profiling genome-wide using uliCUT&RUN in single cells and individual blastocysts. Nat Protoc 16, 2633–2666 (2021).

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