Abstract
Cleavage under targets and release using nuclease (CUT&RUN) allows the chromatin profiling of proteins of interest for which specific antibodies are available. Because it is performed on intact chromatin in situ, CUT&RUN provides exceptional signal over background, making it an ideal choice for chromatin profiling on primary cells available at limited numbers. Here, we describe its application to the profiling of histone post-translational modifications in germ cells isolated from mouse embryos from 12.5 up to 18.5 days postfertilization. This approach can be applied to as low as 100 isolated germ cells, allowing the generation of multiple genome-wide profiles from the cells obtained from a single embryo.
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References
Schmid M, Durussel T, Laemmli UK (2004) ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell 16:147–157. https://doi.org/10.1016/j.molcel.2004.09.007
Skene PJ, Henikoff S (2017) An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6:1–35. https://doi.org/10.7554/eLife.21856
Zhang W, Chen Z, Yin Q et al (2019) Maternal-biased H3K27me3 correlates with paternal-specific gene expression in the human morula. Genes Dev 33:382–387. https://doi.org/10.1101/gad.323105.118
Uyehara CM, McKay DJ (2019) Direct and widespread role for the nuclear receptor EcR in mediating the response to ecdysone in Drosophila. Proc Natl Acad Sci U S A 116:201900343. https://doi.org/10.1073/pnas.1900343116
Ernst C, Eling N, Martinez-Jimenez CP et al (2019) Staged developmental mapping and X chromosome transcriptional dynamics during mouse spermatogenesis. Nat Commun 10:1251. https://doi.org/10.1038/s41467-019-09182-1
Zheng X, Gehring M (2019) Low-input chromatin profiling in Arabidopsis endosperm using CUT&RUN. Plant Reprod 32:63–75. https://doi.org/10.1007/s00497-018-00358-1
Oomen ME, Hansen AS, Liu Y et al (2019) CTCF sites display cell cycle-dependent dynamics in factor binding and nucleosome positioning. Genome Res 29:236–249. https://doi.org/10.1101/gr.241547.118
Park SM, Cho H, Thornton AM et al (2019) IKZF2 drives leukemia stem cell self-renewal and inhibits myeloid differentiation. Cell Stem Cell 24:153–165.e7. https://doi.org/10.1016/j.stem.2018.10.016
Thakur J, Henikoff S (2018) Unexpected conformational variations of the human centromeric chromatin complex. Genes Dev 32:20–25. https://doi.org/10.1101/gad.307736.117
Liu N, Hargreaves VV, Zhu Q et al (2018) Direct promoter repression by BCL11A controls the fetal to adult hemoglobin switch. Cell 173:430–442.e17. https://doi.org/10.1016/j.cell.2018.03.016
Hainer SJ, Bošković A, McCannell KN et al (2019) Profiling of pluripotency factors in single cells and early embryos. Cell 177:1319–1329.e11. https://doi.org/10.1016/j.cell.2019.03.014
Derek Janssens SH (2019) CUT&RUN: targeted in situ genome-wide profiling with high efficiency for low cell numbers. Protocols.io. https://www.protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-zcpf2vn/metadata. Accessed 1 Oct 2019
Zhu Q, Liu N, Orkin SH, Yuan G-C (2019) CUT&RUNTools: a flexible pipeline for CUT&RUN processing and footprint analysis. Genome Biol 20:192. https://doi.org/10.1186/s13059-019-1802-4
Meers MP, Bryson TD, Henikoff JG, Henikoff S (2019) Improved CUT&RUN chromatin profiling tools. Elife 8:e46314. https://doi.org/10.7554/eLife.46314
Skene PJ, Henikoff JG, Henikoff S (2018) Targeted in situ genome-wide profiling with high efficiency for low cell numbers. Nat Protoc 13:1006–1019. https://doi.org/10.1038/nprot.2018.015
Meers MP, Tenenbaum D, Henikoff S (2019) Peak calling by Sparse Enrichment Analysis for CUT&RUN chromatin profiling. Epigenetics Chromatin 12:42. https://doi.org/10.1186/s13072-019-0287-4
Spiller CM, Burnet G, Bowles J (2017) Mouse fetal germ cell isolation and culture techniques. In: Methods in molecular biology. Humana, New York, pp 173–183
Meers MP, Bryson TD, Henikoff JG, Henikoff S (2019) Improved CUT&RUN chromatin profiling tools. Elife 8:1–16. https://doi.org/10.7554/elife.46314
Acknowledgments
We would like to thank François Dossin (Heard lab, EMBL Heidelberg) for advice on CUT&RUN modifications, Martin Möckel (Protein Expression Facility, IMB Mainz) for advice and technical help, and Violeta Morin for comments on the protocol. This work was supported by the Genomics, FACS, and Animal Core Facilities of the Institute of Molecular Biology in Mainz.
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Prakash, S.A., Barau, J. (2021). Chromatin Profiling in Mouse Embryonic Germ Cells by CUT&RUN. In: Ancelin, K., Borensztein, M. (eds) Epigenetic Reprogramming During Mouse Embryogenesis. Methods in Molecular Biology, vol 2214. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0958-3_17
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DOI: https://doi.org/10.1007/978-1-0716-0958-3_17
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