Towards high-throughput parallel imaging and single-cell transcriptomics of microbial eukaryotic plankton.

Area of Science:

• Microbiology
• Eukaryotic cell biology
• Genomics

Background:

• Single-cell transcriptomics offers insights into microbial eukaryotes, but few methods are validated for protists.
• Microbial eukaryotes, or protists, represent a significant portion of plankton and are crucial to marine ecosystems.
• Understanding protist gene expression at the single-cell level is vital for ecological and evolutionary studies.

Purpose of the Study:

• To evaluate the efficacy of microarray single-cell sequencing (MASC-seq) for analyzing protist transcriptomes.
• To adapt and test MASC-seq on diverse planktonic microbial eukaryotes with varying cell structures.
• To assess the potential of MASC-seq for exploring microbial 'dark matter'.

Main Methods:

• Application of MASC-seq technology, which captures transcriptomes alongside cell images.
• Testing MASC-seq on three distinct protist species: Tetrahymena thermophila (ciliate), Phaeodactylum tricornutum (diatom), and Heterocapsa sp. (dinoflagellate).
• Optimization of cell fixation and permeabilization protocols for different protist cell types.

Main Results:

• MASC-seq successfully distinguished single-cell transcripts from background noise in larger protists (ciliate and dinoflagellate).
• Transcriptome expression patterns from MASC-seq correlated well with bulk RNA sequencing data for these larger cells.
• MASC-seq was unable to effectively separate single-cell transcripts from background for smaller diatom cells, indicating a lower signal-to-noise ratio.

Conclusions:

• MASC-seq is a promising technique for single-cell transcriptomic analysis of certain microbial eukaryotes.
• Further methodological optimization is needed to improve the signal-to-noise ratio for smaller cells, like diatoms.
• MASC-seq has the potential to advance the study of under-explored microbial eukaryotes, contributing to understanding 'microbial dark matter'.