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, researchers at the Max Delbrück Center for Molecular Medicine used chemical fixation to address these problems. Methanol fixation allowed them 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, the researchers 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. They 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. The researchers were able to identify diverse cell populations, including neuronal subtypes. As an additional resource, they provide ‘dropbead’, an R package for exploratory data analysis, visualization and filtering of Drop-seq data.
Schematic of experimental workflow
Cultured human (HEK) and mouse (3T3) cells were dissociated, mixed and further processed to analyse the transcriptomes of either live or fixed cells by Drop-seq. Washed cells were gently resuspended in 2 volumes of ice-cold PBS, then fixed by adding 8 volumes of ice-cold methanol. Methanol-fixed cells could be stored for up to several weeks at –80 °C. Prior to Drop-seq, cells were washed before passing them through a 35- to 40-μm cell strainer. Cells were then separately encapsulated in droplets together with a single bead in a microfluidic co-flow device and single-cell transcriptomes sequenced in a highly parallel manner.
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.
Availability – dropbead is freely available for download as an R package: https://github.com/rajewsky-lab/dropbead