Transcriptome Analysis of Sheep IVF embryos based on single Cell RNA-Seq

Abstract Objective: The purpose of the present study was to explore the transcriptome differences of sheep in IVF. Embryos were fertilized in vitro at different developmental stages in order to assess the differences of the function, classification and metabolic pathway of differentially expressed genes and to provide a theoretical basis for revealing the regulatory mechanism of sheep early embryo development. The experiments aimed to promote the development of sheep embryo production technology in vitro. Methods:The single embryos derived from sheep 16-cells and morulae were produced by IVF technology as samples and the sequencing library was constructed by the Smart-Seq2 amplification technology. The transcriber was sequenced by Illumina HiSeqXten high-throughput sequencing technology and the effective sequences were analyzed by functional annotation and related bioinformatic analysis. Results:The results indicated that the Clean reads of 16-cell and morula embryos were 47355570-50855888, of which 93.17%-94.23% reads were compared with the reference genome sequence of sheep; 10 were compared with alternative splicing types of transcription terminal site (TTS) and with the transcription start site (TSS), which accounted for the largest proportion in sheep embryo transcripts. There were 171170-211487 sites of single nucleotide polymorphisms (SNPs) and 9802-10505 sites of novel transcripts in sheep 16-cells and morulas respectively. A total of 10 genes (including novel transcripts) were expressed at the morula stage.The following screening criteria were used: | log2ratio |≥1 and Q-value < 0.05. The differences in the 17,607 DEGs between the 16-cell and morula embryos were significant (370 upregulated and 47 downregulated). From the 16-cell stage to the morula stage, 16,343 DEGs were classified and annotated. It was found that the metabolic processes of BP, CC and MF3 focused on biological functions, such as organic synthesis and nucleic acid synthesis. KEGG analysis indicated 206 pathways that were involved in the 16-cell to morula embryo stage. Two pathways were used for enrichment namely the spliceosome and the signaling pathways regulating pluripotency of stem cells, respectively. Conclusions: The number of differentially expressed genes was identified at different stages of sheep embryo development and the function, classification and metabolic pathway of differentially expressed genes were obtained.