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An optimized protocol for retina single-cell RNA sequencing

ABSTRACT: An optimized protocol for retina single-cell RNA sequencing

PROVIDER: PRJNA643586 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12130643.fastq.gz	Fastqsanger.gz
	SRR12130644.fastq.gz	Fastqsanger.gz
	SRR12130645.fastq.gz	Fastqsanger.gz
	SRR12130646.fastq.gz	Fastqsanger.gz
	SRR12130647.fastq.gz	Fastqsanger.gz

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An optimized protocol for retina single-cell RNA sequencing [scRNA-Seq]

Project description:In this work, we compared different protocols to prepare single-cell suspensions used for scRNAseq and suggest an optimized dissociation protocol for mouse retina, which preserves cell morphology to a higher level leading to an overall increase of gene number per cell. We compared scRNAseq libraries generated with our optimized protocol to publicly available scRNAseq data of mouse retina. We further demonstrate a pipeline to reduce noise in scRNAseq caused by multiplets and ambient RNA.

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Project description:An optimized protocol for retina single-cell RNA sequencing [snRNA-Seq]

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An optimized protocol for retina single-cell RNA sequencing [scRNA-Seq]

Project description:An optimized protocol for retina single-cell RNA sequencing [scRNA-Seq]

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An optimized protocol for retina single-cell RNA sequencing.

Project description:PurposeSingle-cell RNA sequencing (scRNA-seq) is a powerful technique used to explore gene expression at the single cell level. However, appropriate preparation of samples is essential to obtain the most information out of this transformative technology. Generating high-quality single-cell suspensions from the retina is critical to preserve the native expression profile that will ensure meaningful transcriptome data analysis.MethodsWe modified the conditions for rapid and optimal dissociation of retina sample preparation. We also included additional filtering steps in data analysis for retinal scRNA-seq.ResultsWe report a gentle method for dissociation of the mouse retina that minimizes cell death and preserves cell morphology. This protocol also results in detection of higher transcriptional complexity. In addition, the modified computational pipeline leads to better-quality single-cell RNA-sequencing data in retina samples. We also demonstrate the advantages and limitations of using fresh versus frozen retinas to prepare cell or nuclei suspensions for scRNA-seq.ConclusionsWe provide a simple yet robust and reproducible protocol for retinal scRNA-seq analysis, especially for comparative studies.

| S-EPMC7553720 | biostudies-literature

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Project description:Single cell combinatorial indexing RNA sequencing (sci-RNA-seq) is a powerful method for recovering gene expression data from an exponentially scalable number of individual cells or nuclei. However, sci-RNA-seq is a complex protocol that has historically exhibited variable performance on different tissues, as well as lower sensitivity than alternative methods. Here we report a simplified, optimized version of the three-level sci-RNA-seq protocol that is faster, higher yield, more robust, and more sensitive, than the original sci-RNA-seq3 protocol, with reagent costs on the order of 1 cent per cell or less. We showcase the optimized protocol via whole organism analysis of an E16.5 mouse embryo, profiling ~380,000 nuclei in a single experiment. Finally, we introduce a “tiny sci-*” protocol for experiments where input is extremely limited.

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Project description:An optimized protocol for single cell transcriptional profiling by combinatorial indexing

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An optimized protocol for retina single-cell RNA sequencing

Project description:This SuperSeries is composed of the SubSeries listed below.

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