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We have developed a sequencing-based gene expression profiling assay at single-cell resolution by combining a modified single-cell whole transcriptome amplification method with the next generation sequencing technique, the SOLiD™ system. Using this assay, we have shown that blastomeres in a four-cell stage embryo have similar gene expression, which is compatible with the fact that they have similar developmental potential. We proved that compared with cDNA microarray technique, our single-cell cDNA SOLiD sequencing assay can detect expression of thousands of more genes. Moreover, for the genes detected by microarray and SOLiD sequencing, our assay detected new transcript variants for a large proportion of them, which confirms unambiguously at single-cell resolution that the transcriptome complexity is higher than expected traditionally. Finally, by using our assay to Dicer knockout (KO) and Ago2 KO oocytes, we showed that a significant amount of transposons were up-regulated abnormally in Dicer/Ago2 KO mature oocytes compared with wild-type controls.
By analyzing the transcriptome at spectacular and unprecedented depth and accuracy, thousands of new transcript variants/isoforms were found expressed unambiguously in mammalian tissues or organs. These advances greatly accelerate our understanding of the complexity of gene expression regulation and networks for mammalian cells. The technique usually needs milligram amounts of total RNA for analysis, which corresponds to hundreds of thousands of cells. However, under certain conditions, it is practically not possible to get such amounts of materials for analysis, e.g., for early embryonic development. In fact, during mouse early development, when the founder population of germ line primordial germ cells (PGCs) just specified and emerged, there are only approximately 30 PGCs in an embryo. On the other hand, even for in vitro-cultured stem cells, for which the cell amount available for analysis is unlimited, there are limitations. For example, mouse embryonic stem cells, probably the most thoroughly analyzed type of stem cells during the past 27 years, were found to contain multiple subpopulations with strong differences of gene expression and physiological function. Altogether, a more sensitive, next-generation sequencing assay, ideally an assay working to single-cell resolution, is needed for these crucial developmental processes and stem cell biology.
Here, we modified a single-cell whole transcriptome amplification method to make it permissive to amplify cDNAs as long as 3 kb in an efficient and unbiased manner (see Figs. 8 and and9).9). We combined this modified single-cell cDNA amplification method with Applied Biosystems’ next generation sequencing technology, the SOLiD™ system, to set up a single-cell whole transcriptome assay. We proved that it is feasible to obtain gene expression profiles at single-cell resolution, which enables us to ask fundamental biological questions previously not possible, especially in the field of early embryonic development, and to understand biology at the single-cell resolution, which is the uniform, functional unit of any organism.
In summary, we have established a SOLiD sequencing-based gene expression profiling assay at single-cell resolution. We proved that thousands of genes express two or more transcript variants in a same cell. We also proved that in Dicer KO mature oocytes, the transcripts of a lot of transposons and repeat elements are up-regulated abnormally. This single-cell sequencing assay will greatly facilitate understanding the transcriptome complexity during mammalian development, especially in the fields of stem cells and early embryonic development.
We thank Caroline Lee, Melissa A. Barker, Roland Wicki, Cinna Monighetti, Francisco M. De La Vega, and Neil A. Straus for excellent technical help.
Trademarks/Licensing: Applied Biosystems and AB (Design) are registered trademarks, and SOLiD is a trademark of Applied Biosystems or its subsidiaries in the United States and/or certain other countries.