Next-generation sequencing (NGS) has revolutionized genomics, decreasing sequencing costs and allowing researchers to draw correlations between diseases and DNA or RNA changes. Technical advances have enabled the analysis of RNA expression changes between single cells within a heterogeneous population, known as single-cell RNA-seq (scRNA-seq). Despite resolving transcriptomes of cellular subpopulations, scRNA-seq has not replaced RNA-seq, due to higher costs and longer hands-on time. Here, we developed an automated workflow to increase throughput (up to 48 reactions) and to reduce by 75% the hands-on time of scRNA-seq library preparation, using the 10X Genomics Single Cell 3' kit. After gel bead-in-emulsion (GEM) generation on the 10X Genomics Chromium Controller, cDNA amplification was performed, and the product was normalized and subjected to either the manual, standard library preparation method or a fully automated, walk-away method using a Biomek i7 Hybrid liquid handler. Control metrics showed that both quantity and quality of the single-cell gene expression libraries generated were equivalent in size and yield. Key scRNA-seq downstream quality metrics, such as unique molecular identifiers count, mitochondrial RNA content, and cell and gene counts, further showed high correlations between automated and manual workflows. Using the UMAP dimensionality reduction technique to visualize all cells, we were able to further correlate the results observed between the manual and automated methods (R=0.971). The method developed here allows for the fast, error-free, and reproducible multiplex generation of high-quality single-cell gene expression libraries.

中文翻译：

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自动化可实现高通量和可重复的单细胞转录组学文库制备。

下一代测序 (NGS) 彻底改变了基因组学，降低了测序成本，并使研究人员能够得出疾病与 DNA 或 RNA 变化之间的相关性。技术进步使分析异质群体中单个细胞之间的 RNA 表达变化成为可能，称为单细胞 RNA-seq (scRNA-seq)。尽管解决了细胞亚群的转录组，但 scRNA-seq 并没有取代 RNA-seq，因为成本更高且动手时间更长。在这里，我们使用 10X Genomics Single Cell 3' 试剂盒开发了一种自动化工作流程，以提高通量（最多 48 个反应）并减少 75% 的 scRNA-seq 文库制备手动时间。在 10X Genomics Chromium Controller 上生成凝胶珠包乳液 (GEM) 后，进行 cDNA 扩增，对产品进行标准化，并采用手动标准文库制备方法或使用 Biomek i7 Hybrid 液体处理器的全自动无人值守方法。对照指标显示，生成的单细胞基因表达文库的数量和质量在大小和产量上是相当的。关键的 scRNA-seq 下游质量指标，例如唯一分子标识符计数、线粒体 RNA 含量以及细胞和基因计数，进一步显示了自动化和手动工作流程之间的高度相关性。使用 UMAP 降维技术来可视化所有细胞，我们能够进一步关联手动和自动方法之间观察到的结果 (R = 0.971)。这里开发的方法允许快速、无错误、