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Targeted in situ genome-wide profiling with high efficiency for low cell numbers

Abstract

Cleavage under targets and release using nuclease (CUT&RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein–DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&RUN has exceptionally low background levels. CUT&RUN outperforms the most widely used chromatin immunoprecipitation (ChIP) protocols in resolution, signal-to-noise ratio and depth of sequencing required. In contrast to ChIP, CUT&RUN is free of solubility and DNA accessibility artifacts and has been used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here, we present an improved CUT&RUN protocol that does not require isolation of nuclei and provides high-quality data when starting with only 100 cells for a histone modification and 1,000 cells for a transcription factor. From cells to purified DNA, CUT&RUN requires less than a day at the laboratory bench and requires no specialized skills.

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Figure 1: A schematic overview of the CUT&RUN protocol.
Figure 2: TapeStation analysis of an abundant histone epitope (H3K27me3).
Figure 3: CUT&RUN of H3K27me3 requires only 100 cells to profile the human polycomb chromatin landscape.
Figure 4: CUT&RUN requires only 1,000 cells and 4 million fragments to delineate human CTCF peaks.

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Acknowledgements

We thank P. Talbert for helpful comments on the manuscript, C. Codomo for preparing Illumina sequencing libraries and K. Ahmad, A. Spens, N. Thirimanne and R. Resnick, members of our laboratory and colleagues at the Fred Hutchinson Cancer Research Center and elsewhere, who have tried CUT&RUN and provided helpful feedback.

Author information

Authors and Affiliations

Authors

Contributions

P.J.S. and S.H. developed the protocol and performed the experiments. P.J.S., J.G.H. and S.H. analyzed the data. S.H. wrote the manuscript with input from P.J.S. and J.G.H.

Corresponding author

Correspondence to Steven Henikoff.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Tapestation analysis of CUT&RUN cleaved fragments using an anti-CTCF antibody.

The remainder of these samples were used to make libraries for sequencing, with results shown in Figure 4.

Supplementary Figure 2 Recovery of insoluble DNA after release of H3K27me3 DNA into the supernatant.

DNA was extracted from bead pellets of samples used in an experiment profiling H3K27me3 with cell numbers ranging from 50 to 500,000. Concentrations were measured using a Qubit fluorimeter.

Supplementary Figure 3 Spin-column DNA purification partially excludes both large and small fragments.

To test the efficiency of spin columns in binding different length DNA fragments, 2 μg of 10 bp ladder was purified through the column and compared to 2 μg as input. For comparison, 2 μg was phenol extracted and ethanol precipitated. DNA was resolved by 10% polyacrylamide gel electrophoresis and stained with SYBR-gold. Densitometry is shown on the left. For CUT&RUN, removal of large fragments reduces background, but removal of small fragments impacts recovery when profiling DNA-binding proteins. Therefore, spin-column purification (Step 35A) is preferred for nucleosomes, but might be less desirable for transcription factors and very low cell numbers, in which case the alternative PCI protocol (Step 35B) is recommended.

Supplementary Figure 4 Yield increases with digestion time, with little change in signal-to-noise ratio.

By scaling to spike-in DNA, quantitative measurement of the amount of cleaved DNA fragments is possible. The average signal over ~20,000 CTCF CUT&RUN binding sites is compared to an equal number of non-overlapping transcriptional start sites (TSS) as a negative control regions. Spike-in scaled signal was summed over the -50 to +50 bp region relative to the center of the site or TSS.

Supplementary Figure 5 CTCF peak calls show close correspondence between low-cell-number samples.

(A) Percent of intersecting peaks called for the indicated cell number sample, where each entry shows the percent of peaks that intersect for a pair of samples. (B) Heat map representation of the data shown in A on a log2 pixel range scale to illustrate the close correspondence between peak calls over the 1000 to 100,000 cell number range. The low degree of intersection between the ENCODE peaks and all CUT&RUN peaks is largely attributable to the much lower signal-to-noise for ENCODE ChIP-seq. Peaks were called using a threshold method, binning in 50-bp intervals, with a 99.5-%ile threshold of non-zero scores, where maximum peak width = 1500 bp, minimum peak width = 40 bp and interpeak distance = 50 bp.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5. (PDF 560 kb)

Supplementary Methods

Example of a C-shell script for spike-in calibration of CUT&RUN data. (ZIP 1 kb)

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Skene, P., Henikoff, J. & Henikoff, S. Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc 13, 1006–1019 (2018). https://doi.org/10.1038/nprot.2018.015

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