文献_2-2012-Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells.pdfVIP

A rt i c l e s Full-length mrNA-seq from single-cell levels of rNA and individual circulating tumor cells 1,2,7 3,7 4 3 1 1 Daniel Ramsköld , Shujun Luo , Yu-Chieh Wang , Robin Li , Qiaolin Deng , Omid R Faridani , 5 3 4 6 3 Gregory A Daniels , Irina Khrebtukova , Jeanne F Loring , Louise C Laurent , Gary P Schroth Rickard Sandberg1,2 Genome-wide transcriptome analyses are routinely used to monitor tissue-, disease- and cell type–specific gene expression, but it has been technically challenging to generate expression profiles from single cells. Here we describe a robust mRNA-Seq . protocol (Smart-Seq) that is applicable down to single cell levels. Compared with existing methods, Smart-Seq has improved d e read coverage across transcripts, which enhances detailed analyses of alternative transcript isoforms and identification of v r e single-nucleotide polymorphisms. We determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell s e r s transcriptomics by evaluating it on total RNA dilution series. We found that although gene expression estimates from single cells t h have increased noise, hundreds of differentially expressed genes could be identified using few cells per cell type. Applying Smart- g i r Seq to circulating tumor cells from melanomas, we identified distinct gene expression patterns, including candidate biomarkers l l A for melanoma circulating tumor cells. Our protocol will