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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Accession codes
References
Henikoff, S. & Greally, J.M. Epigenetics, cellular memory and gene regulation. Curr. Biol. 26, R644–R648 (2016).
Li, B., Carey, M. & Workman, J.L. The role of chromatin during transcription. Cell 128, 707–719 (2007).
Solomon, M.J. & Varshavsky, A. Formaldehyde-mediated DNA-protein crosslinking: a probe for in vivo chromatin structures. Proc. Natl. Acad. Sci. USA 82, 6470–6474 (1985).
Johnson, D.S., Mortazavi, A., Myers, R.M. & Wold, B. Genome-wide mapping of in vivo protein-DNA interactions. Science 316, 1497–1502 (2007).
Barski, A. et al. High-resolution profiling of histone methylations in the human genome. Cell 129, 823–837 (2007).
Rhee, H.S. & Pugh, B.F. Comprehensive genome-wide protein-DNA interactions detected at single-nucleotide resolution. Cell 147, 1408–1419 (2011).
Skene, P.J. & Henikoff, S. A simple method for generating high-resolution maps of genome-wide protein binding. Elife 4, e09225 (2015).
Kasinathan, S., Orsi, G.A., Zentner, G.E., Ahmad, K. & Henikoff, S. High-resolution mapping of transcription factor binding sites on native chromatin. Nat. Methods 11, 203–209 (2014).
Teytelman, L., Thurtle, D.M., Rine, J. & van Oudenaarden, A. Highly expressed loci are vulnerable to misleading ChIP localization of multiple unrelated proteins. Proc. Natl. Acad. Sci. USA 110, 18602–18607 (2013).
Park, D., Lee, Y., Bhupindersingh, G. & Iyer, V.R. Widespread misinterpretable ChIP-seq bias in yeast. PLoS One 8, e83506 (2013).
Jain, D., Baldi, S., Zabel, A., Straub, T. & Becker, P.B. Active promoters give rise to false positive ′Phantom Peaks′ in ChIP-seq experiments. Nucleic Acids Res. 43, 6959–6968 (2015).
Baranello, L., Kouzine, F., Sanford, S. & Levens, D. ChIP bias as a function of cross-linking time. Chromosome Res. 24, 175–181 (2016).
Meyer, C.A. & Liu, X.S. Identifying and mitigating bias in next-generation sequencing methods for chromatin biology. Nat. Rev. Genet. 15, 709–721 (2014).
Crawford, G.E. et al. Genome-wide mapping of DNase hypersensitive sites using massively parallel signature sequencing (MPSS). Genome Res. 16, 123–131 (2006).
Giresi, P.G., Kim, J., McDaniell, R.M., Iyer, V.R. & Lieb, J.D. FAIRE (formaldehyde-assisted isolation of regulatory elements) isolates active regulatory elements from human chromatin. Genome Res. 17, 877–885 (2007).
Auerbach, R.K. et al. Mapping accessible chromatin regions using Sono-Seq. Proc. Natl. Acad. Sci. USA 106, 14926–14931 (2009).
Kent, N.A., Adams, S., Moorhouse, A. & Paszkiewicz, K. Chromatin particle spectrum analysis: a method for comparative chromatin structure analysis using paired-end mode next-generation DNA sequencing. Nucleic Acids Res. 39, e26 (2011).
Henikoff, J.G., Belsky, J.A., Krassovsky, K., Macalpine, D.M. & Henikoff, S. Epigenome characterization at single base-pair resolution. Proc. Natl Acad. Sci. USA 108, 18318–18323 (2011).
Buenrostro, J.D., Giresi, P.G., Zaba, L.C., Chang, H.Y. & Greenleaf, W.J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins and nucleosome position. Nat. Methods 10, 1213–1218 (2013).
Bernt, K.M. et al. MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L. Cancer Cell 20, 66–78 (2011).
van Steensel, B., Delrow, J. & Henikoff, S. Chromatin profiling using targeted DNA adenine methyltransferase. Nat. Genet. 27, 304–308 (2001).
Schmid, M., Durussel, T. & Laemmli, U.K. ChIC and ChEC; genomic mapping of chromatin proteins. Mol. Cell 16, 147–157 (2004).
Skene, P.J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, e21856 (2017).
Hu, Z. et al. Nucleosome loss leads to global transcriptional up-regulation and genomic instability during yeast aging. Genes Dev. 28, 396–408 (2014).
Orlando, D.A. et al. Quantitative ChIP-Seq normalization reveals global modulation of the epigenome. Cell Rep. 9, 1163–1170 (2014).
Corces, M.R. et al. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat. Genet. 48, 1193–1203 (2016).
Brind′Amour, J. et al. An ultra-low-input native ChIP-seq protocol for genome-wide profiling of rare cell populations. Nat. Commun. 6, 6033 (2015).
Mendoza-Parra, M.A., Pattabhiraman, S. & GRonemeyer, H. Sequential chromatin immunoprecipitation protocol for global analysis through massive parallel sequencing (reChIP-seq). Protoc. Exchange http://dx.doi.org/10.1038/protex.2011.257 (2012).
Lieberman-Aiden, E. et al. Comprehensive mapping of long-range interactions reveals folding principles of the human genome. Science 326, 289–293 (2009).
Tang, Z. et al. CTCF-mediated human 3D genome architecture reveals chromatin topology for transcription. Cell 163, 1611–1627 (2015).
Mumbach, M.R. et al. HiChIP: efficient and sensitive analysis of protein-directed genome architecture. Nat. Methods 13, 919–922 (2016).
Chen, Y. 'TSA-Seq': a novel proximity mapping approach for studying three dimensional genome organization and function. PhD thesis, University of Illinois, http://hdl.handle.net/2142/92935 (2016).
Beagrie, R.A. et al. Complex multi-enhancer contacts captured by genome architecture mapping. Nature 543, 519–524 (2017).
Wahba, L., Costantino, L., Tan, F.J., Zimmer, A. & Koshland, D. S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation. Genes Dev. 30, 1327–1338 (2016).
Sanders, M.M. Fractionation of nucleosomes by salt elution from micrococcal nuclease-digested nuclei. J. Cell Biol. 79, 97–109 (1978).
Davie, J.R. & Saunders, C.A. Chemical composition of nucleosomes among domains of calf thymus chromatin differing in micrococcal nuclease accessibility and solubility properties. J. Biol. Chem. 256, 12574–12580 (1981).
Henikoff, S., Henikoff, J.G., Sakai, A., Loeb, G.B. & Ahmad, K. Genome-wide profiling of salt fractions maps physical properties of chromatin. Genome Res. 19, 460–469 (2009).
Zentner, G.E. & Henikoff, S. High-resolution digital profiling of the epigenome. Nat. Rev. Genet. 15, 814–827 (2014).
Fan, X., Lamarre-Vincent, N., Wang, Q. & Struhl, K. Extensive chromatin fragmentation improves enrichment of protein binding sites in chromatin immunoprecipitation experiments. Nucleic Acids Res. 36, e125 (2008).
Teytelman, L. et al. Impact of chromatin structures on DNA processing for genomic analyses. PLoS One 4, e6700 (2009).
Chung, H.R. et al. The effect of micrococcal nuclease digestion on nucleosome positioning data. PLoS One 5, e15754 (2010).
Zentner, G.E., Kasinathan, S., Xin, B., Rohs, R. & Henikoff, S. ChEC-seq kinetics discriminate transcription factor binding sites by DNA sequence and shape in vivo. Nat. Commun. 6, 8733 (2015).
Liu, X. & Fagotto, F. A method to separate nuclear, cytosolic, and membrane-associated signaling molecules in cultured cells. Sci. Signal. 4, pl2 (2011).
Adam, S.A., Marr, R.S. & Gerace, L. Nuclear protein import in permeabilized mammalian cells requires soluble cytoplasmic factors. J. Cell Biol. 111, 807–816 (1990).
Chen, K. et al. The overlooked fact: fundamental need for spike-in control for virtually all genome-wide analyses. Mol. Cell Biol. 36, 662–667 (2015).
Thorvaldsdottir, H., Robinson, J.T. & Mesirov, J.P. Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration. Brief Bioinform. 14, 178–192 (2013).
Kasinathan, S., Orsi, G.A., Zentner, G.E., Ahmad, K. & Henikoff, S. High-resolution mapping of transcription factor binding sites on native chromatin. Nat. Methods 11, 203–209 (2014).
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
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
Ethics declarations
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)
Rights and permissions
About this article
Cite this article
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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2018.015
This article is cited by
-
Modeling methyl-sensitive transcription factor motifs with an expanded epigenetic alphabet
Genome Biology (2024)
-
Evaluating histone modification analysis of individual preimplantation embryos
BMC Genomics (2024)
-
The chromatin factors SET-26 and HCF-1 oppose the histone deacetylase HDA-1 in longevity and gene regulation in C. elegans
Nature Communications (2024)
-
An epigenetic barrier sets the timing of human neuronal maturation
Nature (2024)
-
Histone lactylation couples cellular metabolism with developmental gene regulatory networks
Nature Communications (2024)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.
