Cell fixation and preservation for droplet-based single-cell transcriptomics.
Jonathan Alles, Nikos Karaiskos, Samantha D Praktiknjo, Stefanie Grosswendt, Philipp Wahle, Pierre-Louis Ruffault, Salah Ayoub, Luisa Schreyer, Anastasiya Boltengagen, Carmen Birchmeier, Robert Zinzen, Christine Kocks, Nikolaus Rajewsky
Author Information
Jonathan Alles: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Nikos Karaiskos: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Samantha D Praktiknjo: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Stefanie Grosswendt: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Philipp Wahle: Systems Biology of Neural Tissue Differentiation, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Pierre-Louis Ruffault: Developmental Biology/Signal Transduction, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Salah Ayoub: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Luisa Schreyer: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Anastasiya Boltengagen: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Carmen Birchmeier: Developmental Biology/Signal Transduction, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Robert Zinzen: Systems Biology of Neural Tissue Differentiation, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany.
Christine Kocks: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany. christine.kocks@mdc-berlin.de. ORCID
Nikolaus Rajewsky: Systems Biology of Gene Regulatory Elements, Berlin Institute for Medical Systems Biology (BIMSB), Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), 13125, Berlin, Germany. rajewsky@mdc-berlin.de.
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.
