Characterization of the single-cell transcriptional landscape by highly multiplex RNA-seq.

Our understanding of the development and maintenance of tissues has been greatly aided by large-scale gene expression analysis. However, tissues are invariably complex, and expression analysis of a tissue confounds the true expression patterns of its constituent cell types. Here we describe a novel strategy to access such complex samples. Single-cell RNA-seq expression profiles were generated, and clustered to form a two-dimensional cell map onto which expression data were projected. The resulting cell map integrates three levels of organization: the whole population of cells, the functionally distinct subpopulations it contains, and the single cells themselves-all without need for known markers to classify cell types. The feasibility of the strategy was demonstrated by analyzing the transcriptomes of 85 single cells of two distinct types. We believe this strategy will enable the unbiased discovery and analysis of naturally occurring cell types during development, adult physiology, and disease.

[1]  L. Pikó,et al.  Patterns of mRNA prevalence and expression of B1 and B2 transcripts in early mouse embryos. , 1987, Development.

[2]  J. Rossant,et al.  Non-injection methods for the production of embryonic stem cell-embryo chimaeras , 1993, Nature.

[3]  W. Schmidt,et al.  CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs. , 1999, Nucleic Acids Research.

[4]  E. Levanon,et al.  Human housekeeping genes are compact. , 2003, Trends in genetics : TIG.

[5]  P. Rorsman,et al.  Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels. , 2005, Genome research.

[6]  R. Singer,et al.  Transcriptional Pulsing of a Developmental Gene , 2006, Current Biology.

[7]  David Bryder,et al.  Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR , 2006, Proceedings of the National Academy of Sciences.

[8]  D. Tranchina,et al.  Stochastic mRNA Synthesis in Mammalian Cells , 2006, PLoS biology.

[9]  Kazuki Kurimoto,et al.  An improved single-cell cDNA amplification method for efficient high-density oligonucleotide microarray analysis , 2006, Nucleic acids research.

[10]  O. R. Borodulina,et al.  Transcripts synthesized by RNA polymerase III can be polyadenylated in an AAUAAA-dependent manner. , 2008, RNA.

[11]  S. Ranade,et al.  Stem cell transcriptome profiling via massive-scale mRNA sequencing , 2008, Nature Methods.

[12]  Eric T. Wang,et al.  Alternative Isoform Regulation in Human Tissue Transcriptomes , 2008, Nature.

[13]  N. Tamamaki,et al.  Method for single-cell microarray analysis and application to gene-expression profiling of GABAergic neuron progenitors , 2008, Neuroscience Research.

[14]  A. Moliner,et al.  Mouse embryonic stem cell-derived spheres with distinct neurogenic potentials. , 2008, Stem cells and development.

[15]  Cole Trapnell,et al.  Ultrafast and memory-efficient alignment of short DNA sequences to the human genome , 2009, Genome Biology.

[16]  B. Williams,et al.  Mapping and quantifying mammalian transcriptomes by RNA-Seq , 2008, Nature Methods.

[17]  Ryoichiro Kageyama,et al.  The cyclic gene Hes1 contributes to diverse differentiation responses of embryonic stem cells. , 2009, Genes & development.

[18]  Catalin C. Barbacioru,et al.  mRNA-Seq whole-transcriptome analysis of a single cell , 2009, Nature Methods.

[19]  Jeffrey G. Reifenberger,et al.  Direct RNA sequencing , 2009, Nature.

[20]  A. Janulaitis,et al.  Novel application of Phi29 DNA polymerase: RNA detection and analysis in vitro and in situ by target RNA-primed RCA. , 2009, RNA.

[21]  Tomoharu Kajiyama,et al.  Quantitative analysis of gene expression in a single cell by qPCR , 2009, Nature Methods.

[22]  Mikael Huss,et al.  Resolution of cell fate decisions revealed by single-cell gene expression analysis from zygote to blastocyst. , 2010, Developmental cell.

[23]  Catalin C. Barbacioru,et al.  Tracing the Derivation of Embryonic Stem Cells from the Inner Cell Mass by Single-Cell RNA-Seq Analysis , 2010, Cell stem cell.