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Straightforward extraction of high-quality RNA from glyoxal-fixed cells facilitates transcriptome analysis after cell staining and sorting by flow cytometry

ABSTRACT: Straightforward extraction of high-quality RNA from glyoxal-fixed cells facilitates transcriptome analysis after cell staining and sorting by flow cytometry

PROVIDER: PRJNA664206 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12664215.fastq.gz	Fastqsanger.gz
	SRR12664216.fastq.gz	Fastqsanger.gz
	SRR12664217.fastq.gz	Fastqsanger.gz
	SRR12664218.fastq.gz	Fastqsanger.gz
	SRR12664219.fastq.gz	Fastqsanger.gz

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Project description:Electronic cell sorting for isolation and culture of dinoflagellates and other marine eukaryotic phytoplankton was compared to the traditional method of manually picking cells using a micropipette. Trauma to electronically sorted cells was not a limiting factor, as fragile dinoflagellates, such as Karenia brevis (Dinophyceae), survived electronic cell sorting to yield viable cells. The rate of successful isolation of large-scale (> 4 litres) cultures was higher for manual picking than for electronic cell sorting (2% vs 0.5%, respectively). However, manual picking of cells is more labor intensive and time consuming. Most manually isolated cells required repicking, as the cultures were determined not to be unialgal after a single round of isolation; whereas, no cultures obtained in this study from electronic single-cell sorting required resorting. A broad flow cytometric gating logic was employed to enhance species diversity. The percentages of unique genotypes produced by manual picking or electronic cell sorting were similar (57% vs 54%, respectively), and each approach produced a variety of dinoflagellate or raphidophyte genera. Alternatively, a highly restrictive gating logic was successfully used to target K. brevis from a natural bloom sample. Direct electronic single-cell sorting was more successful than utilizing a pre-enrichment sort followed by electronic single-cell sorting. The appropriate recovery medium may enhance the rate of successful isolations. Seventy percent of isolated cells were recovered in a new medium (RE) reported here, which was optimized for axenic dinoflagellate cultures. The greatest limiting factor to the throughput of electronic cell sorting is the need for manual postsort culture maintenance and assessment of the large number of isolated cells. However, when combined with newly developed automated methods for growth screening, electronic single-cell sorting has the potential to accelerate the discovery of new algal strains.

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Straightforward extraction of high-quality RNA from glyoxal-fixed cells facilitates transcriptome analysis after cell staining and sorting by flow cytometry

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