Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE209947 | GEO | 2023/05/15

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows

Project description:Background: Single-cell RNA sequencing has been widely adopted to estimate the cellular composition of heterogeneous tissues and obtain transcriptional profiles of individual cells. Multiple approaches for optimal sample dissociation and storage of single cells have been proposed as have single-nuclei profiling methods. What has been lacking is a systematic comparison of their relative biases and benefits. Results: Here, we compare gene expression and cellular composition of single cell suspensions prepared from adult mouse kidney using two tissue dissociation protocols. For each sample we also compare fresh cells to cryopreserved and methanol-fixed cells. Lastly, we compare this single-cell data to that generated using three single-nucleus RNA sequencing workflows. Our data confirms prior reports that digestion on ice avoids the stress response observed with 37°C dissociation. It also reveals cell types more abundant either in the cold or warm dissociations that may represent populations that require gentler or harsher conditions to be released intact. For cell storage, cryopreservation of dissociated cells results in a major loss of epithelial cell types; in contrast, methanol fixation maintains the cellular composition but suffers from ambient RNA leakage. Finally, cell type composition differences are observed between single-cell and single-nucleus RNA sequencing libraries. In particular, we note an under-representation of T, B and NK lymphocytes in the single-nucleus libraries. Conclusions: Systematic comparison of recovered cell types and their transcriptional profiles across the workflows has highlighted protocol-specific biases and thus enables researchers starting single cell experiments to make an informed choice.

2020-05-11 | GSE141115 | GEO

Effect of methanol fixation on single cell RNA sequencing of the murine dentate gyrus (droplet-based scRNAseq).

Project description:We evaluated the effect of methanol (MeOH) fixation on adult murine dentate gyrus (DG) single cell suspensions processed with droplet-based scRNA-seq.

2023-09-22 | GSE241452 | GEO

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

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 | GSE127248 | GEO

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

2019-07-25 | GSE127246 | GEO

Fibroblastic reticular cell lineage convergence in Peyer’s patches governs intestinal homeostasis

Project description:Peyer`s patches (PP) are underpinned by a dense network of FRCs. To elaborate the subset composition and topological organization of PP FRCs, we employed well-characterized Ccl19-Cre and Col6a1-Cre mouse models that permit FRC targeting in secondary lymphoid organs. To define PP FRC subsets and their relation to the observed FRC lineages, we isolated fibroblasts from Col6a1Eyfp and Ccl19Eyfp PPs and performed droplet-based single cell transcriptomics analysis. Taken together, the molecular definition of the PP FRC landscape reveals two FRC lineages furnishing distinct microenvironmental niches.

2021-01-22 | E-MTAB-9162 | biostudies-arrayexpress

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

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

| PRJNA863130 | ENA

Cell fixation and preservation for droplet-based single cell transcriptomics

Project description:Cell fixation and preservation for droplet-based single cell transcriptomics

| PRJNA350512 | ENA

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

Project description:Introduction: The application of single-cell RNA sequencing has greatly improved our under-standing of various cellular and molecular mechanisms involved in physiological and pathophysi-ological processes. However, obtaining living cells for this technique can be difficult under certain conditions. To solve this problem, the methanol fixation method appeared as a promising alternative for routine clinical use. Materials and Methods: In this study, we selected two AML samples that had been fixed in methanol for 12–18 months. Once the cells were rehydrated, these samples were subjected to single-cell RNA sequencing. We then compared the results obtained from these samples with those obtained from the same samples cryopreserved in DMSO. Results: We used a previously validated methanol fixation protocol to perform scRNA-seq on DMSO cryopreserved cells and cells fixed in methanol for more than one year. Preliminary results show that methanol fixation induces some genetic and transcriptional modification compared with DMSO cryopreservation but remains a valuable method for single-cell analysis of primary human leukemia cells. Conclusions: The initial findings from this study highlight certain resemblances in methanol fixation over a 12-month period and cryopreservation with DMSO, along with associated transcriptional level modifications. However, we observed genetic degradation in the fixation condition when extending beyond one year. Despite certain study limitations, it is evident that short-term methanol fixation can be effec-tively used for leukemia blast samples. Its ease of implementation holds the potential to simplify the integration of this technique into routine clinical practice.

2023-08-17 | GSE237239 | GEO

On-capillary cell lysis enables top-down proteomic analysis of single HeLa cells by CE-MS/MS

Project description:Proteomic analysis of limited samples and single cells requires specialized methods that prioritize high sensitivity and minimize sample loss. Consequently, sample preparation is one of the most important steps in limited sample analysis workflows to prevent sample loss. In this work, we have eliminated sample handling and transfer steps by processing intact cells directly in the separation capillary, online with capillary electrophoresis coupled to tandem mass spectrometry (CE-MS/MS) for top-down proteomic (TDP) analysis of low numbers of mammalian cancer cells (<10) and single cells. We assessed spray voltage injection of intact cells from a droplet of cell suspension (~1,000 cells) and demonstrated 0-9 intact cells injected with a dependency on the duration of spray voltage application. Application of spray voltage for 2 min injected an average of 7±2 cells and resulted in 33-57 protein and 40-88 proteoform identifications (N=4). To analyze single cells, manual cell loading by hydrodynamic pressure was used. Replicates of single cells (N=4) lysed on the capillary and analyzed by CE-MS/MS demonstrated a range of 17-40 proteins and 23-50 proteoforms identified. Furthermore, an additional cell line, THP-1, was analyzed at the single-cell level and proteoform abundances were compared to show the capabilities of single-cell top-down proteomics (SC-TDP) for assessing cellular heterogeneity. This study demonstrates the initial application of TDP in single-cell proteome-level profiling. These results represent the highest reported identifications from TDP analysis of a single HeLa cell and prove the tremendous potential for CE-MS/MS on-capillary sample processing for high sensitivity analysis of single cells and limited samples.

2022-10-26 | PXD035339 | Pride

Single Cell and Spatial Analysis of Human Squamous Cell Carcinoma [ST]

Project description:To define the cellular composition and spatial architecture of the tumor micoenvironment, we combined single-cell RNA-sequencing, spatial transcriptomics, and multiplexed ion-beam imaging from 10 patient cutaneous squamous cell carcinoma tumors and site-matched normal skin

2020-06-22 | GSE144239 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data