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

ABSTRACT: High-throughput parallel imaging and single-cell transcriptomics of microbial eukaryotic plankton

PROVIDER: PRJEB56600 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	ERR10355438_1.fastq.gz	Fastqsanger.gz
	ERR10355438_2.fastq.gz	Fastqsanger.gz
	ERR10355439_1.fastq.gz	Fastqsanger.gz
	ERR10355439_2.fastq.gz	Fastqsanger.gz
	ERR10355440_1.fastq.gz	Fastqsanger.gz

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

Project description:In this study we applied MASC-seq (massive and parallel microarray sequencing, https://doi.org/10.1038/ncomms13182), a scRNA-seq method that facilitates sequencing of thousands of cells in parallel, and that couples microscope images with the single cell transcriptome data. For this method, fixed cells are spread over a microarray with 100 μm-sized spots of DNA capture probes with spot-specific indices. The cells are first imaged using a scanning microscope and then permeabilized, releasing their RNA out of the cells and bind to the probes on the array. cDNA is synthesized, harvested and sequenced, and, using the spot-specific barcode-sequences, cDNA sequences stemming from a specific spot (i.e., cell) can be linked to the microscope image of the corresponding cell. However, until now, the MASC-seq method has only been applied to mammalian cells. The aim of this study was to test and adapt the MASC-seq method for application on unicellular eukaryotic plankton. We applied and optimized the method on three cultured plankton representatives, abundant in communities of aquatic environments, Phaeodactylum tricornutum (a diatom, silica and polysaccharide cell walls 23), Heterocapsa sp. (a dinoflagellate, cellulose thecal plates 24) and Tetrahymena thermophila (a ciliate, lipid membrane 25) which all have different size and diverse cell surface structures common to plankton. We optimized several steps in the protocol to make it more suitable for planktonic cells and compared the results from MASC-seq generated single cell transcriptomes to bulk RNA sequencing.

2022-10-26 | E-MTAB-12261 | biostudies-arrayexpress

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Project description:We used single-cell sequencing data and imaging to investigate Eukaryotic plankton from environmental marine samples collected from Coogee, NSW, Australia.

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

Project description: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.

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