Cell fixation and preservation for droplet-based single-cell transcriptomics

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Item Type:

Article

Title:

Cell fixation and preservation for droplet-based single-cell transcriptomics

Creators Name:

Alles, J. and Karaiskos, N. and Praktiknjo, S.D. and Grosswendt, S. and Wahle, P. and Ruffault, P.L. and Ayoub, S. and Schreyer, L. and Boltengagen, A. and Birchmeier, C. and Zinzen, R. and Kocks, C. and Rajewsky, N.

Abstract:

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 altered by stress or ageing. Other challenges are rare cells that need to be collected over several days or samples prepared at different times or locations. METHODS: Here, we used chemical fixation to address these problems. Methanol fixation allowed us to stabilise and preserve dissociated cells for weeks without compromising single-cell RNA sequencing data. RESULTS: By using mixtures of fixed, cultured human and mouse cells, we first 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 cells from dissociated, complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells prepared by fluorescence-activated cell sorting, 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.

Keywords:

Droplet-Based Single-Cell Transcriptomics, Drop-Seq, Gene Expression Pprofiling, Fixation, Alcohol-Based Fixation, Methanol, Fluorescent Activated Cell Sorting (FACS), Primary Cells, Tissue, Animals, Mice

Source:

BMC Biology

ISSN:

1741-7007

Publisher:

BioMed Central

Volume:

Number:

Page Range:

Date:

19 May 2017

Official Publication: