Single-cell sequencing experiments are a new mainstay in biology and have been advancing science especially in the biomedical field. The high pressure to integrate the technology into daily laboratory live requires solid knowledge with respect to potential limitations and precautions to be taken care of before applying it to complex research questions. In the past, we have identified two issues with quality measures neglected by the growing community involving SmartSeq and droplet or micro-well-based scRNASeq methods (1) how to ensure that only single cells are introduced without biasing on light scattering when handling complex cell mixtures and organ preparations or (2) how best to control for (pro-)apoptotic cell contaminations in single-cell sequencing approaches. Sighting of concurrent literature involving single-cell sequencing technologies revealed that these topics are generally neglected or simply approached in silico but not at the bench before generating single-cell data sets. We fear that those important quality aspects are overlooked due to reduced awareness of their importance for guaranteeing the quality of experiments. In this Cytometry rigor issue, we provide experimentally supported guidance on how to circumvent those critical shortcomings in order to promote a better use of the fantastic single-cell sequencing toolbox in biology. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

中文翻译：

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凋亡细胞排除和无偏差单细胞选择是成功单细胞测序应用的重要质量控制要求。

单细胞测序实验是生物学的新支柱，并且一直在推进科学，尤其是在生物医学领域。将该技术集成到日常实验室生活中的高压要求在将其应用于复杂的研究问题之前，需要对潜在的限制和要注意的预防措施有扎实的知识。过去，我们已经确定了两个被日益增长的社区所忽视的质量测量问题，包括 SmartSeq 和基于液滴或微孔的 scRNASeq 方法 (1) 如何确保在处理复杂细胞时只引入单个细胞而不偏向于光散射混合物和器官制剂或 (2) 如何最好地控制单细胞测序方法中的（促）凋亡细胞污染。对涉及单细胞测序技术的并发文献的观察表明，在生成单细胞数据集之前，这些主题通常被忽视或简单地在计算机中处理，而不是在工作台上。我们担心那些重要的质量方面会因为对保证实验质量的重要性认识不足而被忽视。在这个细胞计量学严格问题中，我们提供了有关如何规避这些关键缺点的实验支持指导，以促进在生物学中更好地使用出色的单细胞测序工具箱。© 2019 作者。Cytometry Part A 由 Wiley Periodicals, Inc. 代表 International Society for Advancement of Cytometry 出版。