A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast

  1. Abbas Jariani
  2. Lieselotte Vermeersch
  3. Bram Cerulus
  4. Gemma Perez-Samper
  5. Karin Voordeckers
  6. Thomas Van Brussel
  7. Bernard Thienpont
  8. Diether Lambrechts
  9. Kevin J Verstrepen  Is a corresponding author
  1. VIB-KU Leuven Center for Microbiology, Belgium
  2. VIB-KU Leuven Center for Cancer Biology, Belgium

Abstract

Current methods for single-cell RNA sequencing (scRNA-seq) of yeast cells do not match the throughput and relative simplicity of the state-of-the-art techniques that are available for mammalian cells. In this study, we report how 10x Genomics' droplet-based single-cell RNA sequencing technology can be modified to allow analysis of yeast cells. The protocol, which is based on in-droplet spheroplasting of the cells, yields an order-of-magnitude higher throughput in comparison to existing methods. After extensive validation of the method, we demonstrate its use by studying the dynamics of the response of isogenic yeast populations to a shift in carbon source, revealing the heterogeneity and underlying molecular processes during this shift. The method we describe opens new avenues for studies focusing on yeast cells, as well as other cells with a degradable cell wall.

Data availability

Sequencing data have been deposited in GEO under accession code GSE144820

The following data sets were generated
The following previously published data sets were used

Article and author information

Author details

  1. Abbas Jariani

    VIB Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, Leuven, Belgium
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2715-933X
  2. Lieselotte Vermeersch

    VIB Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, Leuven, Belgium
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5789-2220
  3. Bram Cerulus

    VIB Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, Leuven, Belgium
    Competing interests
    No competing interests declared.
  4. Gemma Perez-Samper

    VIB Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, Leuven, Belgium
    Competing interests
    No competing interests declared.
  5. Karin Voordeckers

    VIB Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, Leuven, Belgium
    Competing interests
    No competing interests declared.
  6. Thomas Van Brussel

    VIB-KU Leuven Laboratory for Translational Genetics, VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium
    Competing interests
    No competing interests declared.
  7. Bernard Thienpont

    VIB-KU Leuven Laboratory for Translational Genetics, VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8772-6845
  8. Diether Lambrechts

    VIB-KU Leuven Laboratory for Translational Genetics, VIB-KU Leuven Center for Cancer Biology, Leuven, Belgium
    Competing interests
    No competing interests declared.
  9. Kevin J Verstrepen

    VIB Laboratory for Systems Biology, VIB-KU Leuven Center for Microbiology, Leuven, Belgium
    For correspondence
    kevin.verstrepen@kuleuven.vib.be
    Competing interests
    Kevin J Verstrepen, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3077-6219

Funding

Fonds Wetenschappelijk Onderzoek

  • Lieselotte Vermeersch
  • Bram Cerulus

Vlaams Instituut voor Biotechnologie

  • Kevin J Verstrepen

European Research Council (Council CoG682009)

  • Kevin J Verstrepen

AB-InBev-Baillet Latour Fund

  • Kevin J Verstrepen

Human Frontier Science Program (246 RGP0050/2013)

  • Kevin J Verstrepen

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Antonis Rokas, Vanderbilt University, United States

Publication history

  1. Received: January 20, 2020
  2. Accepted: May 15, 2020
  3. Accepted Manuscript published: May 18, 2020 (version 1)
  4. Version of Record published: May 29, 2020 (version 2)

Copyright

© 2020, Jariani et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 7,988
    Page views
  • 734
    Downloads
  • 17
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Abbas Jariani
  2. Lieselotte Vermeersch
  3. Bram Cerulus
  4. Gemma Perez-Samper
  5. Karin Voordeckers
  6. Thomas Van Brussel
  7. Bernard Thienpont
  8. Diether Lambrechts
  9. Kevin J Verstrepen
(2020)
A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast
eLife 9:e55320.
https://doi.org/10.7554/eLife.55320

Further reading

    1. Chromosomes and Gene Expression
    2. Developmental Biology
    Lewis Macdonald et al.
    Tools and Resources

    Auxin-inducible degrons are a chemical genetic tool for targeted protein degradation and are widely used to study protein function in cultured mammalian cells. Here we develop CRISPR-engineered mouse lines that enable rapid and highly specific degradation of tagged endogenous proteins in vivo. Most but not all cell types are competent for degradation. By combining ligand titrations with genetic crosses to generate animals with different allelic combinations, we show that degradation kinetics depend upon the dose of the tagged protein, ligand, and the E3 ligase substrate receptor TIR1. Rapid degradation of condensin I and condensin II - two essential regulators of mitotic chromosome structure - revealed that both complexes are individually required for cell division in precursor lymphocytes, but not in their differentiated peripheral lymphocyte derivatives. This generalisable approach provides unprecedented temporal control over the dose of endogenous proteins in mouse models, with implications for studying essential biological pathways and modelling drug activity in mammalian tissues.

    1. Cell Biology
    2. Chromosomes and Gene Expression
    Jakub Gemperle et al.
    Tools and Resources

    CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.