Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing
Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing
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DOI：
10.1038/nprot.2012.022
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发表时间：
2012-04-01
期刊：
影响因子：
14.8
通讯作者：
Linnarsson, Sten
中科院分区：
文献类型：
--
作者：
Islam, Saiful;Kjallquist, Una;Linnarsson, Sten
基因表达的单细胞分析对于复杂组织（包括癌症、发育中的器官以及成体干细胞微环境）的分析日益重要。在此，我们介绍一种通过对mRNA 5'端进行测序来定量分析单细胞基因表达的详细方案。总共裂解96个细胞，并将其mRNA转化为cDNA。利用模板转换机制，在逆转录的同时引入条形码和上游引物结合序列。将所有cDNA混合，然后为5'端测序做准备，包括片段化、接头连接和PCR扩增。这种方法的主要优势在于早期的条形码策略大大降低了成本和时间。与先前的方法相比，它更适合大规模定量分析以及转录起始位点的表征，但不适合检测可变剪接的转录本。样本制备需要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可能还需要4天。
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.