Laboratory for Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Nat Protoc. 2012 Apr 5;7(5):813-28. doi: 10.1038/nprot.2012.022.
Single-cell analysis of gene expression is increasingly important for the analysis of complex tissues, including cancer, developing organs and adult stem cell niches. Here we present a detailed protocol for quantitative gene expression analysis in single cells, by the sequencing of mRNA 5' ends. In all, 96 cells are lysed, and their mRNA is converted to cDNA. By using a template-switching mechanism, a bar code and an upstream primer-binding sequence are introduced simultaneously with reverse transcription. All cDNA is pooled and then prepared for 5' end sequencing, including fragmentation, adapter ligation and PCR amplification. The chief advantage of this approach is the great reduction in cost and time, afforded by the early bar-coding strategy. Compared with previous methods, it is more suitable for large-scale quantitative analysis, as well as for the characterization of transcription start sites, but it is unsuitable for the detection of alternatively spliced transcripts. Sample preparation takes 3 d, and two sets of 96 cells can be prepared in parallel. Finally, the sequencing and data analysis can take an additional 4 d altogether.
单细胞分析基因表达对于分析复杂组织越来越重要，包括癌症、发育器官和成人干细胞龛。在这里，我们提出了一种通过 mRNA 5' 端测序对单细胞进行定量基因表达分析的详细方案。总共裂解 96 个细胞，并将其 mRNA 转化为 cDNA。通过使用模板转换机制，同时引入条形码和上游引物结合序列进行逆转录。所有 cDNA 混合后，然后进行 5' 端测序，包括片段化、接头连接和 PCR 扩增。这种方法的主要优点是早期条形码策略带来的成本和时间的大大减少。与以前的方法相比，它更适合大规模定量分析，以及转录起始位点的特征描述，但不适合检测选择性剪接转录本。样品制备需要 3 天，并且可以并行制备两组 96 个细胞。最后，测序和数据分析总共需要额外 4 天。
