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Characterization of the single-cell (ES and MEF) transcriptional landscape by highly multiplex RNA-Seq

ABSTRACT: 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 constitutent 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 was 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 themselvesâall without need for known markers to classify cell types. The feasibility of the strategy was demonstrated by analyzing the complete transcriptomes of 92 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE29087 | GEO | 2011/05/25

SECONDARY ACCESSION(S): PRJNA140307

REPOSITORIES: GEO

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Project description:Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next-generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis. We investigated gene expression in mouse embryonic cells using microfluidic-facilitated RNA-Seq to analyze 56 single mouse ES cell (mESC) transcriptomes and 6 single mouse embryonic fibroblast (MEF) transcriptomes. To quantitatively evaluate the sensitivity and precision of our technique, we also sequenced 23 libraries from extracted mESC RNA, representing three sets of technical replicates with varying starting amounts of material.

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Characterization of the single-cell (ES and MEF) transcriptional landscape by highly multiplex RNA-Seq

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