Existing protocols for full-length single-cell RNA sequencing produce libraries of high complexity (thousands of distinct genes) with outstanding sensitivity and specificity of transcript quantification. These full-length libraries have the advantage of allowing probing of transcript isoforms, are informative regarding single-nucleotide polymorphisms and allow assembly of the VDJ region of the T- and B-cell-receptor sequences. Since full-length protocols are mostly plate-based at present, they are also suited to profiling cell types where cell numbers are limiting, such as rare cell types during development. A disadvantage of these methods has been the scalability and cost of the experiments, which has limited their popularity as compared with droplet-based and nanowell approaches. Here, we describe an automated protocol for full-length single-cell RNA sequencing, including both an in-house automated Smart-seq2 protocol and a commercial kit–based workflow. The protocols take 3–5 d to complete, depending on the number of plates processed in a batch. We discuss these two protocols in terms of ease of use, equipment requirements, running time, cost per sample and sequencing quality. By benchmarking the lysis buffers, reverse transcription enzymes and their combinations, we have optimized the in-house automated protocol to dramatically reduce its cost. An automated setup can be adopted easily by a competent researcher with basic laboratory skills and no prior automation experience. These pipelines have been employed successfully for several research projects allied with the Human Cell Atlas initiative (www.humancellatlas.org).

现有的全长单细胞 RNA 测序方案产生了高度复杂的文库（数千个不同的基因），具有出色的转录本量化灵敏度和特异性。这些全长文库的优点是允许探测转录亚型，提供有关单核苷酸多态性的信息，并允许组装 T 细胞和 B 细胞受体序列的 VDJ 区域。由于目前全长方案大多是基于板的，因此它们也适用于分析细胞数量有限的细胞类型，例如开发过程中的稀有细胞类型。这些方法的一个缺点是实验的可扩展性和成本，与基于液滴和纳米井的方法相比，这限制了它们的普及。这里，我们描述了全长单细胞 RNA 测序的自动化协议，包括内部自动化 Smart-seq2 协议和基于商业试剂盒的工作流程。该协议需要 3-5 天才能完成，具体取决于批次中处理的板数。我们从易用性、设备要求、运行时间、每个样本的成本和测序质量等方面讨论这两个协议。通过对裂解缓冲液、逆转录酶及其组合进行基准测试，我们优化了内部自动化方案以显着降低其成本。具有基本实验室技能且没有自动化经验的称职研究人员可以轻松采用自动化设置。这些管道已成功用于与人类细胞图谱计划 (www.humancellatlas.org) 相关的几个研究项目。