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Low-Input CUT&RUN for Mouse Oocytes and Preimplantation Embryos.
Ryoya Hayashi1,2, Azusa Inoue3,4
1Laboratory for Epigenome Inheritance, RIKEN Center for Integrative Medical Sciences, Yokohama, Kanagawa, Japan.
Methods in Molecular Biology (Clifton, N.J.)
|September 29, 2022
Summary
We optimized the CUT&RUN assay for fragile cells like mouse oocytes. This modified protocol prevents cell damage and chromatin loss, enabling successful low-input DNA analysis.
Area of Science:
- Molecular Biology
- Genomics
- Epigenetics
Background:
- Cleavage Under Target & Release Using Nuclease (CUT&RUN) is a low-input method for detecting protein-DNA interactions.
- Standard CUT&RUN protocols can fail with fragile cells, such as mouse oocytes, due to cell damage and chromatin loss.
Purpose of the Study:
- To develop an optimized low-input CUT&RUN protocol for mouse oocytes and preimplantation embryos.
- To overcome limitations of existing CUT&RUN methods when applied to fragile cell types.
Main Methods:
- A modified CUT&RUN protocol was developed, completing antibody and protein A-MNase binding before cell attachment to magnetic beads.
- Cells were bound to Concanavalin A-coated magnetic beads after key binding steps to prevent mechanical stress.
Main Results:
- The optimized protocol prevents the crushing of fragile oocytes and early embryos.
- Unwanted chromatin loss during CUT&RUN procedures is significantly reduced.
- Successful application of CUT&RUN to low-input mouse oocyte and preimplantation embryo samples was achieved.
Conclusions:
- This modified CUT&RUN protocol enhances the analysis of protein-DNA interactions in fragile cells.
- The method provides a robust approach for low-input epigenomic studies in early developmental stages.
- This technique is crucial for understanding gene regulation in sensitive biological samples.
