PBMC Fixation and Processing for Chromium Single-Cell RNA Sequencing
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PROVIDER: PRJNA449292 | ENA |
REPOSITORIES: ENA
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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:BackgroundInterest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells.MethodsThe methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10× Chromium platform.ResultsWhen methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to 3-month storage steps. Resuspension but not rehydration in 3× 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 3× SSC were successfully implemented into 10× 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 also worked in several other fixed primary cell types and cell lines as in live ones.ConclusionsWe expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.
| S-EPMC6050658 | biostudies-literature
Project description:In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMC) was assessed by isolating and stabilizing PBMC-derived RNA from three individuals either immediately after phlebotomy or following a 4 hour delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133plus 2.0 GeneChip®. Comparison of gene expression levels (p<0.05 and ≥ 2-fold expression change) identified 327 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 hours. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis. Keywords: Technical comparison
2011-03-22 | GSE21039 | GEO
Project description:Single-cell RNA Sequencing data from PBMC subjected to whole blood processing delay
| PRJNA672733 | ENA
Project description:Single-cell RNA Sequencing data from PBMC subjected to whole blood processing delay
| PRJNA672734 | ENA
Project description:In the analysis of peripheral blood gene expression, timely processing of samples is essential to ensure that measurements reflect in vivo biology, rather than ex vivo sample processing variables. The effect of processing delays on global gene expression patterns in peripheral blood mononuclear cells (PBMC) was assessed by isolating and stabilizing PBMC-derived RNA from three individuals either immediately after phlebotomy or following a 4 hour delay. RNA was labeled using NuGEN Ovation labeling and probed using the Affymetrix HG U133plus 2.0 GeneChip®. Comparison of gene expression levels (p<0.05 and ? 2-fold expression change) identified 327 probe sets representing genes with increased expression and 46 indicating decreased expression after 4 hours. The trends in expression patterns associated with delayed processing were also apparent in an independent set of 276 arrays of RNA from human PBMC samples with varying processing times. These data indicate that the time between sample acquisition, initiation of processing, and when the RNA is stabilized should be a prime consideration when designing protocols for translational studies involving PBMC gene expression analysis. Keywords: Technical comparison 3 subjects, 3 samples each. First sample is prepared immediately, 2nd after 2 hour delay and last after a 4 hour delay.
2011-03-22 | E-GEOD-21039 | biostudies-arrayexpress
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:The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.
2024-03-11 | GSE229944 | 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:Processing of NODULE INCEPTION controls transition to nitrogen fixation in root nodules
| PRJNA737413 | ENA