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FACS Isolation of Viable Cells in Different Cell Cycle Stages from Asynchronous Culture for RNA Sequencing

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Cellular Heterogeneity

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1745))

Abstract

Recently developed high-throughput analytical techniques (e.g., protein mass spectrometry and nucleic acid sequencing) allow unprecedentedly sensitive, in-depth studies in molecular biology of cell proliferation, differentiation, aging, and death. However, the initial population of asynchronous cultured cells is highly heterogeneous by cell cycle stage, which complicates immediate analysis of some biological processes. Widely used cell synchronization protocols are time-consuming and can affect the finely tuned biochemical pathways leading to biased results. Besides, certain cell lines cannot be effectively synchronized. The current methodological challenge is thus to provide an effective tool for cell cycle phase-based population enrichment compatible with other required experimental procedures. Here, we describe an optimized approach to live cell FACS based on Hoechst 33342 cell-permeable DNA-binding fluorochrome staining. The proposed protocol is fast compared to traditional synchronization methods and yields reasonably pure fractions of viable cells for further experimental studies including high-throughput RNA-seq analysis.

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Acknowledgments

The work was supported by the Moscow State University Program of Development and by the Russian Science Foundation (grant #14-14-00088, sequencing and data analysis; grant #14-50-00029, optimization of RNA extraction and library preparation). Daria M. Potashnikova and Sergey A. Golyshev have contributed equally to this work.

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Author notes

  1. Daria M. Potashnikova and Sergey A. Golyshev have contributed equally to this chapter.

Authors and Affiliations

  1. Department of Cell Biology and Histology, School of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia

    Daria M. Potashnikova

  2. A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, Moscow, Russia

    Sergey A. Golyshev, Alexey A. Penin, Maria D. Logacheva, Anna V. Klepikova & Eugene V. Sheval

  3. Department of Genetics, School of Biology, M.V. Lomonosov Moscow State University, Moscow, Russia

    Alexey A. Penin

  4. Institute fzòr Information Transmission Problems, Russian Academy of Sciences, Moscow, Russia

    Alexey A. Penin, Maria D. Logacheva, Anna V. Klepikova & Andrey A. Mironov

  5. School of Bioengineering and Bioinformatics, M.V. Lomonosov Moscow State University, Moscow, Russia

    Anna V. Klepikova, Anastasia A. Zharikova & Andrey A. Mironov

  6. Department of Biology, School of Sciences and Technology, Nazarbayev University, Astana, Kazakhstan

    Ivan A. Vorobjev

Authors
  1. Daria M. Potashnikova
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  2. Sergey A. Golyshev
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  3. Alexey A. Penin
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  4. Maria D. Logacheva
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  5. Anna V. Klepikova
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  6. Anastasia A. Zharikova
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  7. Andrey A. Mironov
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  8. Eugene V. Sheval
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  9. Ivan A. Vorobjev
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Corresponding author

Correspondence to Ivan A. Vorobjev .

Editor information

Editors and Affiliations

  1. PCMM-Harvard Medical School, Boston Children’s Hospital, Boston, Massachusetts, USA

    Natasha S. Barteneva

  2. Department of Biology, School of Sciences and Technology, Nazarbayev University, Astana, Kazakhstan

    Ivan A. Vorobjev

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Potashnikova, D.M. et al. (2018). FACS Isolation of Viable Cells in Different Cell Cycle Stages from Asynchronous Culture for RNA Sequencing. In: Barteneva, N., Vorobjev, I. (eds) Cellular Heterogeneity. Methods in Molecular Biology, vol 1745. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7680-5_18

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  • DOI: https://doi.org/10.1007/978-1-4939-7680-5_18

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7679-9

  • Online ISBN: 978-1-4939-7680-5

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