Abstract
Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Algorithms
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Animals
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Argonaute Proteins
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Blastomeres / cytology
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Blastomeres / metabolism*
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Cyclin E / genetics
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DEAD-box RNA Helicases / genetics
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DNA, Complementary / chemical synthesis
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DNA, Complementary / genetics
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Databases, Nucleic Acid
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Endoribonucleases / genetics
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Eukaryotic Initiation Factor-2 / genetics
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Female
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Gene Expression Profiling / methods*
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Kruppel-Like Transcription Factors / genetics
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Mice
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Mice, Inbred Strains
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Mice, Knockout
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Mice, Transgenic
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Oligonucleotide Array Sequence Analysis
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Oocytes / metabolism*
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Polymerase Chain Reaction / methods
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Protein Isoforms / genetics
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Proteins / genetics
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RNA, Messenger / analysis
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RNA, Messenger / metabolism
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Ribonuclease III
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Sequence Alignment
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Sequence Analysis, DNA / methods*
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Up-Regulation / genetics
Substances
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Ago2 protein, mouse
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Argonaute Proteins
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Cyclin E
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DNA, Complementary
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Dppa5 protein, mouse
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Eukaryotic Initiation Factor-2
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Klf2 protein, mouse
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Kruppel-Like Transcription Factors
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Protein Isoforms
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Proteins
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RNA, Messenger
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Endoribonucleases
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Dicer1 protein, mouse
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Ribonuclease III
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DEAD-box RNA Helicases