Abstract
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.
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Acknowledgements
We thank P. Sekyrova (Karolinska Institutet) for providing cultured cells. This work was supported by grants from the Swedish Foundation for Strategic Research (MDB09-0052) and from the European Research Council (261063).
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S.I. developed the method and performed optimization experiments, analyzed data and co-wrote the manuscript; U.K. performed initial experiments; A.M. performed cell culture and FACS sorting; P.Z. performed experiments to optimize the method; J.-B.F. performed sequencing experiments and advised on method development; P.L. developed the bioinformatics pipeline and analyzed data; S.L. conceived of the method, supervised the development, developed the bioinformatics pipeline and co-wrote the manuscript.
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S.L. has applied for patents covering parts of the method.
Supplementary information
Supplementary Tables 1 and 2
Supplementary table 1: List of Template Switching Oligos (DOCX 134 kb)
Supplementary table 2: Orientation of the template switching oligo in 96 well plate
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Islam, S., Kjällquist, U., Moliner, A. et al. Highly multiplexed and strand-specific single-cell RNA 5′ end sequencing. Nat Protoc 7, 813–828 (2012). https://doi.org/10.1038/nprot.2012.022
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DOI: https://doi.org/10.1038/nprot.2012.022
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