Cell fixation and preservation for droplet-based single-cell transcriptomics.
Cell fixation and preservation for droplet-based single-cell transcriptomics.
复制标题
DOI：
10.1186/s12915-017-0383-5
复制
发表时间：
2017-05-19
期刊：
影响因子：
5.4
通讯作者：
Rajewsky N
中科院分区：
文献类型：
--
作者：
Alles J;Karaiskos N;Praktiknjo SD;Grosswendt S;Wahle P;Ruffault PL;Ayoub S;Schreyer L;Boltengagen A;Birchmeier C;Zinzen R;Kocks C;Rajewsky N
基于液滴的微流体技术的最新进展使得能够以定量、高度并行且具有成本效益的方式对数千个单个细胞进行转录谱分析。一个关键且常常具有限制性的步骤是制备处于未受干扰状态的细胞，这些细胞不因应激或衰老而改变。其他挑战包括需要在数天内收集的稀有细胞，以及在不同时间或地点制备的样本。
在此，我们利用化学固定来解决这些问题。甲醇固定使我们能够在不损害单细胞RNA测序数据的情况下，将解离的细胞稳定并保存数周。
通过使用固定的培养的人类和小鼠细胞混合物，我们首先表明单个转录组能够可靠地归属于两个物种中的一个。来自活细胞和固定样本的单细胞基因表达与大量mRNA - seq数据具有良好的相关性。然后我们将甲醇固定应用于对来自解离的复杂组织的原代细胞进行转录谱分析。来自果蝇胚胎的低RNA含量细胞，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、储存和单细胞液滴RNA - seq之后都得到了成功分析。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Drop - seq数据的探索性数据分析、可视化和过滤的R软件包。
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的机会。
本文的网络版（doi:10.1186/s12915 - 017 - 0383 - 5）包含补充材料，授权用户可获取。
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. The online version of this article (doi:10.1186/s12915-017-0383-5) contains supplementary material, which is available to authorized users.