FACS isolation of viable cells in different cell cycle stages from asynchronous culture for RNA sequencing

Daria M. Potashnikova, Sergey A. Golyshev, Alexey A. Penin, Maria D. Logacheva, Anna V. Klepikova, Anastasia A. Zharikova, Andrey A. Mironov, Eugene V. Sheval, Ivan Vorobyev

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages315-335
Number of pages21
DOIs
Publication statusPublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1745
ISSN (Print)1064-3745

Keywords

  • Cell cycle
  • FACS
  • Flow cytometry
  • Hoechst 33342
  • Population heterogeneity
  • RNA-seq

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Potashnikova, D. M., Golyshev, S. A., Penin, A. A., Logacheva, M. D., Klepikova, A. V., Zharikova, A. A., Mironov, A. A., Sheval, E. V., & Vorobyev, I. (2018). FACS isolation of viable cells in different cell cycle stages from asynchronous culture for RNA sequencing. In Methods in Molecular Biology (pp. 315-335). (Methods in Molecular Biology; Vol. 1745). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-7680-5_18