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

Abstract

Single-cell transcriptomics has the potential to provide novel insights into poorly studied microbial eukaryotes. Although several such technologies are available and benchmarked on mammalian cells, few have been tested on protists. Here, we applied a microarray single-cell sequencing (MASC-seq) technology, that generates microscope images of cells in parallel with capturing their transcriptomes, on three species representing important plankton groups with different cell structures; the ciliate Tetrahymena thermophila, the diatom Phaeodactylum tricornutum, and the dinoflagellate Heterocapsa sp. Both the cell fixation and permeabilization steps were adjusted. For the ciliate and dinoflagellate, the number of transcripts of microarray spots with single cells were significantly higher than for background spots, and the overall expression patterns were correlated with that of bulk RNA, while for the much smaller diatom cells, it was not possible to separate single-cell transcripts from background. The MASC-seq method holds promise for investigating "microbial dark matter”, although further optimizations are necessary to increase the signal-to-noise ratio.

Revealing gene expression heterogeneity in a clonal population of Tetrahymena thermophila through single-cell RNA sequencing

“…Single-cell RNA sequencing (scRNA-seq) provides a powerful tool to dissect the mechanisms behind this variability [ [3] , [4] , [5] ]. While mammalian cells have been extensively analyzed using scRNA-seq, studies on ciliates, particularly those involving sequencing thousands of cells, have been comparatively rare, despite previous studies employing bulk RNA-seq or scRNA-seq with approximately 550 single cells of ciliates [ [6] , [7] , [8] , [9] , [10] ]. One reason for this disparity is the larger size of ciliate cells (typically ∼500 μm in length) compared to mammalian cells.…”

Revealing Gene Expression Heterogeneity in a Clonal Population ofTetrahymena thermophilathrough Single-Cell RNA Sequencing

“…Single-cell RNA sequencing (scRNA-seq) provides a powerful tool to dissect the mechanisms behind this variability (Saliba et al, 2014; Buettner et al, 2015; Kuchina et al, 2021). While mammalian cells have been extensively analyzed using scRNA-seq, studies on ciliates, particularly those involving sequencing thousands of cells, have been comparatively rare, despite previous studies employing bulk RNA-seq or scRNA-seq with approximately 550 single cells of ciliates (Xiong et al, 2012; Kolisko et al, 2014; Yan et al, 2019; Yang et al, 2023; Grujčić et al, 2024). One reason for this disparity is the larger size of ciliate cells (typically ∼500 μm in length) compared to mammalian cells.…”