Alles Jonathan, Karaiskos Nikos, Praktiknjo Samantha D, Grosswendt Stefanie, Wahle Philipp, Ruffault Pierre-Louis, Ayoub Salah, Schreyer Luisa, Boltengagen Anastasiya, Birchmeier Carmen, Zinzen Robert, Kocks Christine, Rajewsky Nikolaus
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.
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.
BMC Biol. 2017 May 19;15(1):44. doi: 10.1186/s12915-017-0383-5.
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 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.
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.
基于微滴的微流控技术的最新进展使得以定量、高度平行且经济高效的方式对数千个单个细胞进行转录谱分析成为可能。一个关键且常常具有局限性的步骤是将细胞制备成未受干扰的状态，即不受应激或老化影响。其他挑战包括需要在数天内收集的稀有细胞，或在不同时间或地点制备的样本。
在这里，我们使用化学固定来解决这些问题。甲醇固定使我们能够稳定并保存解离后的细胞数周，而不会影响单细胞RNA测序数据。
通过使用固定的、培养的人类和小鼠细胞混合物，我们首先表明可以可靠地将单个转录组归为这两个物种之一。活细胞样本和固定细胞样本的单细胞基因表达与大量mRNA测序数据相关性良好。然后，我们将甲醇固定应用于解离的复杂组织中的原代细胞的转录谱分析。果蝇胚胎中RNA含量低的细胞，以及通过荧光激活细胞分选制备的小鼠后脑和小脑细胞，在固定、储存和单细胞微滴RNA测序后成功进行了分析。我们能够识别出不同的细胞群体，包括神经元亚型。作为一项额外的资源，我们提供了“dropbead”，这是一个用于探索性数据分析、可视化和过滤Drop-seq数据的R包。
我们预计，一种简单的细胞固定方法的可用性将在多种生物学背景下为以单细胞分辨率分析转录动态开辟许多新机会。
