A new protocol for single-cell RNA-seq reveals stochastic gene expression during lag phase in budding yeast
- VIB-KU Leuven Center for Microbiology, Belgium
- 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
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Transition between fermentation and respiration determines historydependent behavior in fluctuating carbon sourcesNCBI Gene Expression Omnibus, , GSE116246.
Article and author information
Author details
Lieselotte Vermeersch
Kevin J Verstrepen
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.
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.
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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
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The mRNA 5'-cap structure removal by the decapping enzyme DCP2 is a critical step in gene regulation. While DCP2 is the catalytic subunit in the decapping complex, its activity is strongly enhanced by multiple factors, particularly DCP1, which is the major activator in yeast. However, the precise role of DCP1 in metazoans has yet to be fully elucidated. Moreover, in humans, the specific biological functions of the two DCP1 paralogs, DCP1a and DCP1b, remain largely unknown. To investigate the role of human DCP1, we generated cell lines that were deficient in DCP1a, DCP1b, or both to evaluate the importance of DCP1 in the decapping machinery. Our results highlight the importance of human DCP1 in decapping process and show that the EVH1 domain of DCP1 enhances the mRNA-binding affinity of DCP2. Transcriptome and metabolome analyses outline the distinct functions of DCP1a and DCP1b in human cells, regulating specific endogenous mRNA targets and biological processes. Overall, our findings provide insights into the molecular mechanism of human DCP1 in mRNA decapping and shed light on the distinct functions of its paralogs.
