Abstract
Chromatin immunoprecipitation (ChIP) is a technique that has been widely used to interrogate DNA–protein interactions in cells. In recent years, human pluripotent stem cell (hPSC)-derived 3D organoids have emerged as a powerful model to understand human development and diseases. Performing ChIP in hPSC-derived 3D organoids is a useful approach to dissect the roles of transcription factors or co-factors and to understand the epigenetic landscape in human development and diseases. However, performing ChIP in 3D organoids is more challenging than monolayer cultures, and an optimized protocol is needed for interpretable data. Hence, in this chapter, we describe in detail a protocol for performing ChIP in hPSC-derived islet-like cells as an example, from organoid harvest to ChIP-qPCR data analysis. This chapter also highlights potential pitfalls and provides recommendations for troubleshooting.
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Acknowledgments
The authors thank members of the Teo laboratory for the critical reading of this manuscript. W.X.T. is supported by the NUS Research Scholarship. N.H.J.N. is supported by NMRC OFYIRG18may040 and the first A*STAR Career Development Award (CDA). A.K.K.T. is supported by the Institute of Molecular and Cell Biology (IMCB), A*STAR, NMRC Open Fund-Young Individual Research Grant (OF-YIRG) OFYIRG16may014, A*STAR ETPL Gap Funding ETPL/18-GAP005-R20H, Lee Foundation Grant SHTX/LFG/002/2018, Skin Innovation Grant SIG18011, NMRC OF-LCG/DYNAMO, FY2019 SingHealth Duke-NUS Surgery Academic Clinical Programme Research Support Programme Grant, Precision Medicine and Personalised Therapeutics Joint Research Grant 2019, Industry Alignment Fund – Industry Collaboration Project (IAF-ICP) I1901E0049 and the second A*STAR-AMED Joint Grant Call 192B9002.
Declaration of interests: N.H.J.N. and A.K.K.T. are founders and shareholders of BetaLife Pte Ltd.
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Tan, W.X., Bok, C.M., Ng, N.H.J., Teo, A.K.K. (2022). Chromatin Immunoprecipitation in Human Pluripotent Stem Cell-Derived 3D Organoids to Analyze DNA–Protein Interactions. In: Kannan, N., Beer, P. (eds) Stem Cell Assays. Methods in Molecular Biology, vol 2429. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1979-7_14
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DOI: https://doi.org/10.1007/978-1-0716-1979-7_14
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