Abstract
Cleavage Under Target & Release Using Nuclease (CUT&RUN) enables the detection of DNA regions that are bound by a protein of interest. This method is suitable for low-input materials because of the absence of an immunoprecipitation step. However, it sometimes fails when applying it to fragile cells, such as mouse oocytes. Here we describe our low-input CUT&RUN protocol optimized for mouse oocyte and preimplantation embryo samples in which the primary antibody and protein A-MNase binding steps are completed before the cells are bound to Concanavalin A-coated magnetic beads. This modification prevents crush of oocytes and early embryos and unwanted loss of chromatin during CUT&RUN procedures.
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Acknowledgments
We thank Dr. Steven Henikoff for providing the protein A-MNase and Drs. Weikun Xia and Wei Xie for helpful discussion on CUT&RUN. This project was partly supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT) Leading Initiative for Excellent Young Researchers Grant to A.I., Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for Scientific Research (KAKENHI) (18H02359 to A.I.), Grant-in-Aid for Scientific Research on Innovative Areas (19H05754 to A.I.), Grant-in-Aid for JSPS Fellows (20J21541 to R.H.), Japan Agency for Medical Research and Development PRIME (JP18gm6110012 and 21gm6310022h0001 to A.I.), Uehara Memorial Foundation to A.I., and RIKEN Pioneering Project “Genome Building from TADs” to A.I.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Hayashi, R., Inoue, A. (2023). Low-Input CUT&RUN for Mouse Oocytes and Preimplantation Embryos. In: Hatada, I., Horii, T. (eds) Epigenomics. Methods in Molecular Biology, vol 2577. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2724-2_6
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DOI: https://doi.org/10.1007/978-1-0716-2724-2_6
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Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2723-5
Online ISBN: 978-1-0716-2724-2
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