Full-length RNA-seq from single cells using Smart-seq2
Full-length RNA-seq from single cells using Smart-seq2
复制标题
DOI：
10.1038/nprot.2014.006
复制
发表时间：
2014-01-01
期刊：
影响因子：
14.8
通讯作者：
Sandberg, Rickard
中科院分区：
文献类型：
--
作者：
Picelli, Simone;Faridani, Omid R.;Sandberg, Rickard
精确定量单个细胞中基因表达的新方法有望揭示细胞间变异的程度、功能和起源。不同的单细胞RNA-seq高通量方法在覆盖范围、灵敏度和多路复用能力上有所不同。我们最近引入了Smart-seq用于单细胞转录组分析，并随后对方法进行了优化，以提高灵敏度、准确性和转录本全长覆盖范围。在这里，我们提出了Smart-seq2的详细方案，允许使用标准试剂生成全长cDNA和测序文库。从细胞挑选到最终的库准备测序，整个过程大约需要2个小时；测序将需要额外的1-3天，这取决于策略和测序器。目前的限制是缺乏链特异性和无法检测非聚腺苷化（polyA(-)） RNA。
Emerging methods for the accurate quantification of gene expression in individual cells hold promise for revealing the extent, function and origins of cell-to-cell variability. Different high-throughput methods for single-cell RNA-seq have been introduced that vary in coverage, sensitivity and multiplexing ability. We recently introduced Smart-seq for transcriptome analysis from single cells, and we subsequently optimized the method for improved sensitivity, accuracy and full-length coverage across transcripts. Here we present a detailed protocol for Smart-seq2 that allows the generation of full-length cDNA and sequencing libraries by using standard reagents. The entire protocol takes similar to 2 d from cell picking to having a final library ready for sequencing; sequencing will require an additional 1-3 d depending on the strategy and sequencer. The current limitations are the lack of strand specificity and the inability to detect nonpolyadenylated (polyA(-)) RNA.