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Distinguishing States of Arrest: Genome-Wide Descriptions of Cellular Quiescence Using ChIP-Seq and RNA-Seq Analysis

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Cellular Quiescence

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1686))

Abstract

Regenerative potential in adult stem cells is closely associated with the establishment of—and exit from—a temporary state of quiescence. Emerging evidence not only provides a rationale for the link between lineage determination programs and cell cycle regulation but also highlights the understanding of quiescence as an actively maintained cellular program, encompassing networks and mechanisms beyond mitotic inactivity or metabolic restriction. Interrogating the quiescent genome and transcriptome using deep-sequencing technologies offers an unprecedented view of the global mechanisms governing this reversibly arrested cellular state and its importance for cell identity. While many efforts have identified and isolated pure target stem cell populations from a variety of adult tissues, there is a growing appreciation that their isolation from the stem cell niche in vivo leads to activation and loss of hallmarks of quiescence. Thus, in vitro models that recapitulate the dynamic reversibly arrested stem cell state in culture and lend themselves to comparison with the activated or differentiated state are useful templates for genome-wide analysis of the quiescence network.

In this chapter, we describe the methods that can be adopted for whole genome epigenomic and transcriptomic analysis of cells derived from one such established culture model where mouse myoblasts are triggered to enter or exit quiescence as homogeneous populations. The ability to synchronize myoblasts in G0 permits insights into the genome in “deep quiescence.” The culture methods for generating large populations of quiescent myoblasts in either 2D or 3D culture formats are described in detail in a previous chapter in this series (Arora et al. Methods Mol Biol 1556:283–302, 2017). Among the attractive features of this model are that genes isolated from quiescent myoblasts in culture mark satellite cells in vivo (Sachidanandan et al., J Cell Sci 115:2701–2712, 2002) providing a validation of its approximation of the molecular state of true stem cells. Here, we provide our working protocols for ChIP-seq and RNA-seq analysis, focusing on those experimental elements that require standardization for optimal analysis of chromatin and RNA from quiescent myoblasts, and permitting useful and revealing comparisons with proliferating myoblasts or differentiated myotubes.

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Acknowledgments

We gratefully acknowledge Divya Tej Sowpati for help with the bioinformatics analysis pipeline and development of custom visualization tools. HG was supported by doctoral fellowships from CSIR. The Mishra and Dhawan labs are supported by core funds from the Council of Scientific and Industrial Research to CCMB, and a collaborative grant from the Dept. of Biotechnology Indo-Australia Biotechnology Fund. JD also acknowledges funding from and the Dept. of Biotechnology Indo-Danish Strategic Fund, Indo-French Center for the Promotion of Advanced Research and from the Dept. of Biotechnology Institute for Stem Cell Biology and Regenerative Medicine.

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Authors and Affiliations

  1. CSIR-Centre for Cellular and Molecular Biology, Habsiguda, Uppal Road, Hyderabad, 500007, India

    Surabhi Srivastava, Hardik P. Gala, Rakesh K. Mishra & Jyotsna Dhawan

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  1. Surabhi Srivastava
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  2. Hardik P. Gala
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  3. Rakesh K. Mishra
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  4. Jyotsna Dhawan
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Correspondence to Jyotsna Dhawan .

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  1. Texas Children’s Hospital, Baylor College of Medicine, Houston, Texas, USA

    H. Daniel Lacorazza

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Srivastava, S., Gala, H.P., Mishra, R.K., Dhawan, J. (2018). Distinguishing States of Arrest: Genome-Wide Descriptions of Cellular Quiescence Using ChIP-Seq and RNA-Seq Analysis. In: Lacorazza, H. (eds) Cellular Quiescence. Methods in Molecular Biology, vol 1686. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7371-2_16

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  • DOI: https://doi.org/10.1007/978-1-4939-7371-2_16

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