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DMSO cryopreservation: the method of choice for droplet-based single-cell RNA sequencing of preserved cells [Drop-Seq]

ABSTRACT: DMSO cryopreservation: the method of choice for droplet-based single-cell RNA sequencing of preserved cells [Drop-Seq]

PROVIDER: PRJNA524475 | ENA |

REPOSITORIES: ENA

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DMSO cryopreservation: the method of choice for droplet-based single-cell RNA sequencing of preserved cells [Drop-Seq]

Project description:Recently, combining single-cell RNA sequencing (scRNA-seq) with upstream cell preservation procedures such as cryopreservation or methanol fixation has become more common. By separating cell handling and preparation, from downstream library generation, scRNA-seq workflows are more flexible and manageable. However, the inherent transcriptomics changes associated with cell preservation and how they may bias further downstream analysis remain unknown. Here, we present a side-by-side droplet-based scRNA-seq analysis, comparing the gold standard – fresh cells – to three different cell preservation workflows: dimethyl sulfoxide based cryopreservation, methanol fixation and CellCover reagent. Cryopreservation proved to be the most robust protocol, maximizing both cell integrity and low background ambient RNA. Importantly, gene expression profiles from fresh cells correlated most with those of cryopreserved cells. Such similarities were consistently observed across the tested cell lines (R ≥ 0.97), monocyte-derived macrophages (R = 0.97) and immune cells (R = 0.99). In contrast, both methanol fixation and CellCover preservation showed an increased ambient RNA background and an overall lower gene expression correlation to fresh cells. Thus, our results demonstrate the superiority of cryopreservation over other cell preservation methods. We expect our comparative study to provide single cell omics researchers invaluable support when integrating cell preservation into their scRNA-seq studies.

2019-07-25 | GSE127246 | GEO

DMSO cryopreservation: the method of choice for droplet-based single-cell RNA sequencing of preserved cells

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DMSO cryopreservation: the method of choice for droplet-based single-cell RNA sequencing of preserved cells [10X Genomics]

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Drop-Seq analysis of ERCC spike in capture

Project description:A bead supsension and a solution of ERCC spike-ins at a concentration of ~100,000 molecules per droplet was used in Drop-Seq, a novel technology for high-throughput single cell mRNAseq An estimated 84 beads were selected for amplification.

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Methanol fixation is the method of choice for droplet-based single-cell transcriptomics of neural cells

Project description:Single-cell transcriptomics methods have become very popular to study the cellular composition of organs and tissues and characterize the expression profiles of the individual cells that compose them. The main critical step for single-cell transcriptomics methods is sample preparation. Several methods have been developed to preserve cells after sample dissociation to uncouple sample handling from library preparation. Yet, the suitability of these methods depends on the types of cells to be processed. In this project, we perform a systematic comparison of preservation methods for droplet-based single-cell RNA-seq (scRNA-seq) on human neural progenitor cell populations derived from induced pluripotent stem cells (iPSCs) and highlight their strengths and weaknesses. We compared the cellular composition and expression profile of single-cell suspensions from fresh NPCs with that of NPCs preserved with Dimethyl Sulfoxide (DMSO), Methanol, vivoPHIX and Acetil-methanol (ACME). Our results show that while DMSO provides the highest cell quality in terms of RNA molecules and genes detected per cell. Yet, it strongly affects the cellular composition and the expression profile of the resulting datasets. In contrast, methanol fixed samples display a cellular composition like that of fresh samples while providing a good cell quality and smaller expression biases. Taken together, our results show that methanol fixation is the method of choice for performing droplet-based single-cell transcriptomics experiments on neural cell populations.

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DMSO cryopreservation: the method of choice for droplet-based single-cell RNA sequencing of preserved cells

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

2019-07-25 | GSE127249 | GEO

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Project description:Drop-BS: high-throughput single-cell bisulfite seqeuncing on a microfluidic droplet platform

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