文献_Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing.pdfVIP

protocol Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing 1 1 2 1 3 1 Saiful Islam , Una Kjällquist , Annalena Moliner , Pawel Zajac , Jian-Bing Fan , Peter Lönnerberg Sten Linnarsson1 1 2 Laboratory for Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. 3Illumina Inc., San Diego, California, USA. Correspondence should be addressed to S.L. (sten.linnarsson@ki.se). Published online 5 April 2012; 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 d