Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom.
Francis Crick Institute, London, United Kingdom.
Nat Commun. 2023 Aug 8;14(1):4788. doi: 10.1038/s41467-023-40322-w.
Droplet microfluidic methods have massively increased the throughput of single-cell sequencing campaigns. The benefit of scale-up is, however, accompanied by increased background noise when processing challenging samples and the overall RNA capture efficiency is lower. These drawbacks stem from the lack of strategies to enrich for high-quality material or specific cell types at the moment of cell encapsulation and the absence of implementable multi-step enzymatic processes that increase capture. Here we alleviate both bottlenecks using fluorescence-activated droplet sorting to enrich for droplets that contain single viable cells, intact nuclei, fixed cells or target cell types and use reagent addition to droplets by picoinjection to perform multi-step lysis and reverse transcription. Our methodology increases gene detection rates fivefold, while reducing background noise by up to half. We harness these properties to deliver a high-quality molecular atlas of mouse brain development, despite starting with highly damaged input material, and provide an atlas of nascent RNA transcription during mouse organogenesis. Our method is broadly applicable to other droplet-based workflows to deliver sensitive and accurate single-cell profiling at a reduced cost.
液滴微流控方法极大地提高了单细胞测序工作的通量。然而，随着规模的扩大，在处理具有挑战性的样本时，背景噪声增加，整体 RNA 捕获效率降低。这些缺点源于缺乏在细胞封装时富集高质量材料或特定细胞类型的策略，以及缺乏可实施的增加捕获的多步酶处理过程。在这里，我们使用荧光激活液滴分选来缓解这两个瓶颈，富集包含单个活细胞、完整核、固定细胞或目标细胞类型的液滴，并通过皮升级注射向液滴中添加试剂，以执行多步裂解和逆转录。我们的方法将基因检测率提高了五倍，同时将背景噪声降低了一半。我们利用这些特性，即使从高度受损的输入材料开始，也能提供高质量的小鼠大脑发育分子图谱，并提供小鼠器官发生过程中新生 RNA 转录的图谱。我们的方法广泛适用于其他基于液滴的工作流程，以降低成本实现敏感和准确的单细胞分析。
