Ludwig Institute for Cancer Research, Stockholm, Sweden.
1] Ludwig Institute for Cancer Research, Stockholm, Sweden. [2] Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
Nat Protoc. 2014 Jan;9(1):171-81. doi: 10.1038/nprot.2014.006. Epub 2014 Jan 2.
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 ∼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.
新兴的方法可用于准确量化单个细胞中的基因表达，有望揭示细胞间变异性的程度、功能和起源。不同的高通量单细胞 RNA-seq 方法在覆盖度、灵敏度和多重检测能力上存在差异。我们最近引入了 Smart-seq 用于单个细胞的转录组分析，随后对该方法进行了优化，以提高灵敏度、准确性和跨转录本的全长覆盖度。本文详细介绍了 Smart-seq2 的实验方案，该方案使用标准试剂生成全长 cDNA 和测序文库。从细胞挑取到最终文库准备测序，整个方案大约需要 2 天时间；测序还需要额外的 1-3 天，具体取决于测序策略和测序仪。目前的限制是缺乏链特异性，以及无法检测非多聚腺苷酸化（polyA(-)）RNA。
