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Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

ABSTRACT: Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

PROVIDER: PRJNA499073 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8129710_1.fastq.gz	Fastqsanger.gz
	SRR8129710_2.fastq.gz	Fastqsanger.gz
	SRR8129703.fastq.gz	Fastqsanger.gz
	SRR8129704.fastq.gz	Fastqsanger.gz
	SRR8129705_1.fastq.gz	Fastqsanger.gz

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Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes

Project description:We provide a method for high quality RNA purification out of a small number (5000 - 100,000) of FACS sorted zebrafish cells followed by RNA sequencing

2019-03-11 | GSE121917 | GEO

Purification of high-quality RNA from a small number of fluorescence activated cell sorted zebrafish cells for RNA sequencing purposes.

Project description:BackgroundTransgenic zebrafish lines with the expression of a fluorescent reporter under the control of a cell-type specific promoter, enable transcriptome analysis of FACS sorted cell populations. RNA quality and yield are key determinant factors for accurate expression profiling. Limited cell number and FACS induced cellular stress make RNA isolation of sorted zebrafish cells a delicate process. We aimed to optimize a workflow to extract sufficient amounts of high-quality RNA from a limited number of FACS sorted cells from Tg(fli1a:GFP) zebrafish embryos, which can be used for accurate gene expression analysis.ResultsWe evaluated two suitable RNA isolation kits (the RNAqueous micro and the RNeasy plus micro kit) and determined that sorting cells directly into lysis buffer is a critical step for success. For low cell numbers, this ensures direct cell lysis, protects RNA from degradation and results in a higher RNA quality and yield. We showed that this works well up to 0.5× dilution of the lysis buffer with sorted cells. In our sort settings, this corresponded to 30,000 and 75,000 cells for the RNAqueous micro kit and RNeasy plus micro kit respectively. Sorting more cells dilutes the lysis buffer too much and requires the use of a collection buffer. We also demonstrated that an additional genomic DNA removal step after RNA isolation is required to completely clear the RNA from any contaminating genomic DNA. For cDNA synthesis and library preparation, we combined SmartSeq v4 full length cDNA library amplification, Nextera XT tagmentation and sample barcoding. Using this workflow, we were able to generate highly reproducible RNA sequencing results.ConclusionsThe presented optimized workflow enables to generate high quality RNA and allows accurate transcriptome profiling of small populations of sorted zebrafish cells.

| S-EPMC6425699 | biostudies-literature

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The Extracellular RNA Quality Control (exRNAQC) study: RNA Exome sequencing of RNA purification kit study (exRNAQC004)

2021-05-10 | E-MTAB-10504 | biostudies-arrayexpress

The Extracellular RNA Quality Control (exRNAQC) study: Small RNA sequencing of RNA purification kit study (exRNAQC011)

Project description:To evaluate the impact of the RNA purification method on extracellular RNA (exRNA) sequencing, 8 different RNA purification kits were compared by applying Small RNA sequencing (Illumina) to exRNA from human healthy donor plasma. Minimum and maximum plasma input volumes recommended by the manufacturers were tested in triplicate. Due to donor privacy concerns the raw data for this study have been submitted to the controlled-access archive EGA under the accession EGAS00001005263.

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Large particle fluorescence-activated cell sorting enables high quality single cell RNA-sequencing and functional analysis of adult cardiomyocytes

Project description:The use of single cell RNA sequencing (scRNA-seq) remains limited in cardiac pathology owing to technical difficulties associated with the isolation of single adult cardiomyocytes (CMs). Here, we investigated the capability of large-particle fluorescence-activated cell sorting (LP-FACS) for isolation of viable single adult CMs. We found that LP-FACS readily outperforms conventional FACS for isolation of structurally competent CMs, including large CMs. Additionally, LP-FACS enables isolation of fluorescent CMs from mosaic models. Importantly, the sorted CMs allow generation of high-quality scRNA-seq libraries. Unlike CMs isolated via previously utilized fluidic or manual methods, LP-FAC-isolated CMs generate libraries exhibiting normal levels of mitochondrial transcripts. Moreover, LP-FACS isolated CMs remain functionally competent and can be studied for contractile properties.

2019-07-02 | GSE133640 | GEO

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Project description:High-speed fluorescence image-enabled cell sorting

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Protocol for the purification and transcriptomic analysis of mouse astrocytes using GFAT

Project description:Astrocytes are glial cells of the central nervous system that modulate neuronal function. Here, we present glyoxal-fixed astrocyte nuclei transcriptomics (GFAT), a protocol for the purification and transcriptomic analysis of astrocyte nuclei from the cortex and cerebellum of adult and aged fresh mouse brain. We describe steps for tissue dissection, glyoxal fixation, homogenization, nuclei isolation, antibody staining, fluorescence-activated cell sorting, and RT-qPCR or bulk RNA sequencing. GFAT does not require transgenic lines or viral injection and allows parallel astrocyte and neuron profiling.

2023-09-15 | GSE233509 | GEO

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