转录组
固定(群体遗传学)
生物
细胞
计算生物学
核糖核酸
细胞生物学
单细胞分析
活体细胞成像
基因
基因表达
遗传学
作者
Jonathan Alles,Nikos Karaiskos,Samantha D. Praktiknjo,Stefanie Grosswendt,Philipp Wahle,Pierre-Louis Ruffault,Salah Ayoub,Luisa Schreyer,Anastasiya Boltengagen,Carmen Birchmeier,Robert P. Zinzen,Christine Kocks,Nikolaus Rajewsky
摘要
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 compromised by stress or ageing. Another challenge are rare cells that need to be collected over several days, or samples prepared at different times or locations. Results Here, we used chemical fixation to overcome these problems. Methanol fixation allowed us to stabilize and preserve dissociated cells for weeks. By using mixtures of fixed human and mouse cells, we 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 single cells from dissociated complex tissues. Low RNA content cells from Drosophila embryos, as well as mouse hindbrain and cerebellum cells sorted by FACS, 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.
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