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

ABSTRACT: 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.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE127248 | GEO | 2019/07/25

REPOSITORIES: GEO

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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

Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation

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2023-08-17 | GSE237239 | GEO

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2018-07-25 | GSE112845 | GEO

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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2023-09-22 | GSE241452 | GEO

Effect of Cryopreservation on Autologous Chimeric Antigen Receptor T Cell Characteristics

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2020-10-03 | GSE158929 | GEO

Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation

Project description:Single-Cell Transcriptome Analysis of Acute Myeloid Leukemia Cells Using Methanol Fixation and Cryopreservation

| PRJNA994456 | ENA

Single-cell transcriptome of cerebral organoids with and without methanol fixation

Project description:To study how methanol fixation affects single-cell transcriptomic measurement, two cerebral organoids were dissociated. Cell suspension of each organoid was split into two aliquots. Methanol fixation was applied to one of the two aliquots. Single-cell RNA-seq with 10x Genomics was applied to the two aliquots separately.

2020-08-31 | E-MTAB-9473 | biostudies-arrayexpress

An optimized workflow for transcriptomic analysis from archival paraformaldehyde-fixed retinal tissues collected by laser capture microdissection

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