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

BMC Biology
Jonathan AllesNikolaus Rajewsky

Abstract

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. 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. 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...Continue Reading

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Methods Mentioned

BETA
PCR
Drop-seq
single-cell sequencing
Chip
fluorescence-activated cell so rting
FACS
flow cytometry
cDNA library construction
by flow cytometry

Software Mentioned

InDrop
ImaGO )
Seurat
tSNE
Ensembl
R
seq
Drop
dropbead
DigitalExpression

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