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Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells

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Stem Cell Heterogeneity

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1516))

Abstract

Mouse Embryonic Stem cells (mESCs) show heterogeneous and dynamic expression of important pluripotency regulatory factors. Single-cell analysis has revealed the existence of cell-to-cell variability in the expression of individual genes in mESCs. Understanding how these heterogeneities are regulated and what their functional consequences are is crucial to obtain a more comprehensive view of the pluripotent state.

In this chapter we describe how to analyze transcriptional heterogeneity by monitoring gene expression of Nanog, Oct4, and Sox2, using single-molecule RNA FISH in single mESCs grown in different cell culture medium. We describe in detail all the steps involved in the protocol, from RNA detection to image acquisition and processing, as well as exploratory data analysis.

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Acknowledgments

This work was supported by Fundação para a Ciência e Tecnologia, Portugal [SFRH/ BPD/78313/2011 to E.A., SFRH/BD/80191/2011 to A.M.V.G. and PTDC/SAUOBD/100664/2008].

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Authors and Affiliations

  1. DHenrique Lab, Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Av. Prof. Egas Moniz, Lisbon, 1649-028, Portugal

    Ana M. V. Guedes, Domingos Henrique & Elsa Abranches

  2. Instituto de Histologia e Biologia do Desenvolvimento, Lisbon, Portugal

    Ana M. V. Guedes, Domingos Henrique & Elsa Abranches

Authors
  1. Ana M. V. Guedes
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  2. Domingos Henrique
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  3. Elsa Abranches
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Corresponding author

Correspondence to Elsa Abranches .

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Editors and Affiliations

  1. Ottawa Hospital Research Institute, Ottawa, Ontario, Canada

    Kursad Turksen

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© 2016 Springer Science+Business Media New York

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Guedes, A.M.V., Henrique, D., Abranches, E. (2016). Dissecting Transcriptional Heterogeneity in Pluripotency: Single Cell Analysis of Mouse Embryonic Stem Cells. In: Turksen, K. (eds) Stem Cell Heterogeneity. Methods in Molecular Biology, vol 1516. Humana Press, New York, NY. https://doi.org/10.1007/7651_2016_356

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  • DOI: https://doi.org/10.1007/7651_2016_356

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6549-6

  • Online ISBN: 978-1-4939-6550-2

  • eBook Packages: Springer Protocols

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