Effect of methanol fixation on single-cell RNA sequencing data
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE150993 | GEO | 2021/05/18
REPOSITORIES: GEO
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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
Project description:Background: Interest in single-cell whole transcriptome analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works, investigators have used live cells which represent several inconveniences and limitations. Some recent cell fixation methods did not work with most primary cells including immune cells. Methods: The methanol-fixation and new processing method was introduced to preserve PBMCs for single-cell RNA sequencing (scRNA-Seq) analysis on 10X Chromium platform. Results: When methanol fixation protocol was broken up into three steps, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to three-month storage steps. Resuspension but not rehydration in 3X saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3X SSC were successfully implemented into 10X Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol was validated in CD8+ T cell and several other cell types. Conclusions: We expect that the methanol-based cell fixation procedure presented here will substantially enable complex experimental design with primary cells at single cell resolution.
2018-07-25 | GSE112845 | GEO
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
Project description:We evaluated the effect of methanol (MeOH) fixation on adult murine dentate gyrus (DG) single cell suspensions.
2023-09-22 | GSE232052 | GEO
Project description:Background: Recent developments in droplet-based microfluidics allow the transcriptional profiling of thousands of individual cells, in a quantitative, highly parallel and cost-effective way. A critical, often limiting step is the preparation of cells in an unperturbed state, not compromised by stress or ageing. Another challenge are rare cells that need to be collected over several days, or samples prepared at different times or locations. Results: Here, we used chemical fixation to overcome these problems. Methanol fixation allowed us to stabilize and preserve dissociated cells for weeks. By using mixtures of fixed human and mouse cells, we showed that individual transcriptomes could be confidently assigned to one of the two species. Single-cell gene expression from live and fixed samples correlated well with bulk mRNA-seq data. We then applied methanol fixation to transcriptionally profile primary single cells from dissociated complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells sorted by FACS, were successfully analysed after fixation, storage and single-cell droplet RNA-seq. We were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, we provide 'dropbead', an R package for exploratory data analysis, visualization and filtering of Drop-seq data. Conclusions: We expect that the availability of a simple cell fixation method will open up many new opportunities in diverse biological contexts to analyse transcriptional dynamics at single cell resolution.
2017-05-03 | GSE89164 | GEO
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
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.
2023-05-15 | GSE209947 | GEO
Project description:Effect of methanol fixation on single-cell RNA sequencing data
| PRJNA634342 | ENA
Project description:Single-cell transcriptome of cerebral organoids with and without methanol fixation
| PRJEB39985 | ENA
Project description:Archival formalin-fixed paraffin-embedded (FFPE) tissue samples hold a wealth of transcriptomic information; however, little is known about potential artifacts. Previously, we identified a consistent shift in global RNA-sequencing profiles between matching frozen and FFPE samples. We hypothesized that this shift was from fixing fresh tissue in formalin. To test this idea, RNA-sequencing was performed on liver samples collected from male mice treated with 600 ppm of a reference chemical (phenobarbital, 600 ppm phenobarbital) or vehicle control for 7 days. Samples were divided into: (1) fresh-frozen (FR); (2) directly fixed in 10% buffered formalin for 18 hours followed by paraffin embedding (FFPE); (3) frozen then fixed as FFPE (FR>FFPE); or (4) frozen then fixed in 70% ethanol followed by paraffin embedding (FR>OH) (n=6/group/condition). Direct fixation resulted in 2946 differentially expressed genes (DEGs), 98% of which were down-regulated. Freezing prior to fixation resulted in ≥95% fewer DEGs vs. FR, indicating that most formalin-derived transcriptional effects occurred with fixation. This was supported by follow-up studies, which identified consistent enrichment in oxidative stress, mitochondrial dysfunction, and transcription elongation pathways with formalin fixation. Notably, formalin fixation in the parent study did not significantly impact chemical response profiles, which were consistent with CAR/PXR activation and 600 ppm phenobarbital exposure. Our results demonstrate distinct transcriptional effects of formalin fixation that could impact gene expression studies using FFPE samples.
2020-09-08 | GSE148174 | GEO