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Effect of methanol fixation on single-cell RNA sequencing data

ABSTRACT: Effect of methanol fixation on single-cell RNA sequencing data

PROVIDER: PRJNA634342 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11826200_1.fastq.gz	Fastqsanger.gz
	SRR11826200_2.fastq.gz	Fastqsanger.gz
	SRR11826300_1.fastq.gz	Fastqsanger.gz
	SRR11826300_2.fastq.gz	Fastqsanger.gz
	SRR11826400_1.fastq.gz	Fastqsanger.gz

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Effect of methanol fixation on single-cell RNA sequencing data

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Effect of methanol fixation on single cell RNA sequencing of the murine dentate gyrus.

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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.

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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.

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Effect of methanol fixation on single cell RNA sequencing of the murine dentate gyrus.

Project description:Effect of methanol fixation on single cell RNA sequencing of the murine dentate gyrus.

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PBMC Fixation and Processing for Chromium Single-Cell RNA Sequencing

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.

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