Single-cell Transcriptome Anaylsis Of Two-cell Stage Mouse Embryos

High-throughout sequencing (also deep sequencing) is a new biological research technology has been rapid development in recent years. And it has been widely used in epigenetics, genomics and transcriptomics. The traditional method for transcriptome analysis requires at least103cells. It is difficult to obtain such a high-purity sample that contains only one type of cell, because organism has many types of cells, and each cell type has different physiological function and phenotype. With common methods, it is difficult to analyze the samples of small number of cells, such as early embryos, Heterogeneous tissues and tumors. The single-cell transcriptome analysis techniques based on the second generation sequencing technology made us analyze genome-wide expression profiling at single cell level. It also helps us to identify characteristic expression feature of specific cell types and find new cell types. And it resolves the problem to analyze genome-wide expression profiling of small number of cells from the fundamental.This study is based on the SOLiD sequencing system, with two-cell stage murine embryos as research materials, researches the trascriptome at single-cell level.1. We use microscope to isolate cells from two-stage embryos. And we contrast the sequencing library. Then we assess the quality of the library by workflow analysis (WFA). The WFA report shows that the quality of library is suitable for sequencing.2. We sequence the library base on the SOLiD sequencing system. After sequencing, we count the number of reads and evaluate the quality of the reads. The numbers of reads derive from samples are as follows:E234.5million; E338.9million; E4-138.6million; E4-237.5million.3. The numbers of the mapping reads are more than6.2million. And the quality scores are greater than30. The results show that our sequencing results are realizable.4. Correlation analysis shows that the expression profiles of four samples are very similar. Pearson correlation coefficient between E2and E3is0.98, and between E4-1and E4-2is0.97. The results show gene expression profiles of the samples from two different two-cell stage embryos and from a same two-stage cell embryo are very similar.5. Difference analysis shows there are1337significant different genes and29070no significant different genes between two different two-cell stage embryos. And there are1337significant different genes and29122no significant different genes between the samples from a same two-cell stage embryo.6. GO enrichment analysis shows expression profiles of two-cell stage embryos are mainly associated with cell proliferation, including cell cycle, RNA processing, cell division.Through the above researches, we have established a single-cell transcriptome detection technology. We got two-cell stage mouse embryos transcriptome, and verified its reliability and authenticity. We have laid the foundations for the research work of next step.