Dataset Information

PBMC fixation and processing for Chromium single-cell RNA sequencing.

ABSTRACT:

Background

Interest 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.

Methods

The 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.

Results

When 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.

Conclusions

We 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.

SUBMITTER: Chen J

PROVIDER: S-EPMC6050658 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Publications

PBMC fixation and processing for Chromium single-cell RNA sequencing.

Chen Jinguo J Cheung Foo F Shi Rongye R Zhou Huizhi H Lu Wenrui W

Journal of translational medicine 20180717 1

<h4>Background</h4>Interest 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 cel ...[more]

PMID: 30016977

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Project description:Technological advances in transcriptome sequencing of single cells provided an unprecedented view on tissue composition and cellular heterogeneity. While several studies have compared different single cell RNA-seq methods with respect to data quality and their ability to distinguish cellular subpopulations, none of these comparative studies investigated the heterogeneity of the cellular transcriptional response upon a chemical perturbation. In this study, we evaluated the transcriptional response of NGP neuroblastoma cells upon nutlin-3 treatment using the C1, ddSeq and Chromium single cell systems. These systems and library preparation methods are representative for the wide variety of platforms, ranging from microfluids chips to droplet-based systems and from full transcript sequencing to 3’ end sequencing. In parallel, we used bulk RNA-seq for molecular characterization of the transcriptional response. Two complementary metrics to evaluate performance were applied: the first is the number and identification of differentially expressed genes as robustly assessed by two statistical models, and the second is enrichment analysis of biological signals, which is independent of sample size or number of cells evaluated. Where relevant, we downsampled sequencing library size, selected cell subpopulations based on specific RNA abundance features, or created pseudobulk samples to make the data more comparable. While the C1 detects the highest number of genes per cell and better resembles bulk RNA-seq, the Chromium identifies most differentially expressed genes, albeit still substantially fewer than bulk RNA-seq. Gene set enrichment analyses reveals that detection of the most abundant genes in single cell RNA-seq experiments is sufficient for molecular phenotyping. Finally, single cell RNA-seq reveals a heterogeneous response of NGP cells upon nutlin-3 treatment, pinpointing putative late-responders or resistant cells, hidden in bulk RNA-seq experiments.

Dataset Information

PBMC fixation and processing for Chromium single-cell RNA sequencing.

Background

Methods

Results

Conclusions

Publications

PBMC fixation and processing for Chromium single-cell RNA sequencing.

Similar Datasets

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