Sanada Chad D, Ooi Aik T
Fluidigm Corporation, South San Francisco, CA, USA.
Methods Mol Biol. 2019;1979:185-195. doi: 10.1007/978-1-4939-9240-9_12.
Single-cell functional analysis provides a natural next step in the now widely adopted single-cell mRNA sequencing studies. Functional studies can be designed to study cellular context by using single-cell culture, perturbation, manipulation, or treatment. Here we present a method for a functional study of 48 single cells by single-cell isolation, dosing, and mRNA sequencing with an integrated fluidic circuit (IFC) on the Fluidigm Polaris™ system. The major procedures required to execute this protocol are (1) cell preparation and staining; (2) priming, single-cell selection, cell dosing, cell staining, and cDNA generation on the Polaris IFC; and (3) preparation and sequencing of single-cell mRNA-seq libraries. The cell preparation and staining steps employ the use of a universal tracking dye to trace all cells that enter the IFC, while additional fluorescence dyes chosen by the user can be used to differentiate cell types in the overall mix. The steps on the Polaris IFC follow standard protocols, which are also described in the Fluidigm user documentation. The library preparation step adds Illumina Nextera XT indexes to the cDNA generated on the Polaris IFC. The resulting sequencing libraries can be sequenced on any Illumina sequencing platform.
单细胞功能分析是目前广泛采用的单细胞mRNA测序研究的自然延伸。功能研究可以通过单细胞培养、扰动、操作或处理来设计，以研究细胞环境。在这里，我们介绍一种在Fluidigm Polaris™系统上通过单细胞分离、加样和使用集成流体回路（IFC）进行mRNA测序来对48个单细胞进行功能研究的方法。执行此方案所需的主要步骤包括：（1）细胞制备和染色；（2）在Polaris IFC上进行预充、单细胞选择、细胞加样、细胞染色和cDNA生成；以及（3）单细胞mRNA-seq文库的制备和测序。细胞制备和染色步骤使用通用追踪染料来追踪所有进入IFC的细胞，而用户选择的其他荧光染料可用于区分总体混合物中的细胞类型。Polaris IFC上的步骤遵循标准方案，Fluidigm用户文档中也有描述。文库制备步骤将Illumina Nextera XT索引添加到在Polaris IFC上生成的cDNA中。所得的测序文库可在任何Illumina测序平台上进行测序。
