Next-generation sequencing Facilitates Quantitative Analysis of Wild Type and EED-/- mice cerebellum Transcriptomes and Genome-wide maps of chromatin state in Wild Type and EED-/- mice cerebellum

Purpose: The goals of this study are to compare Wild Type and EED-/- cerebellum transcriptome profiling (RNA-seq) to quantitative reverse transcription polymerase chain reaction (qRT-PCR) differentially expressed genes and find target genes. We performed chromatin-immunoprecipitation coupled to sequencing (ChIP-seq) to dissect the relationship between the differentially expressed genes and the occupancy of H3K27me3, H3K27ac and EED. Methods: Cerebellum mRNA profiles of 14-day-old (P14) wild-type (WT) and Embryonic Ectoderm Development conditional knockout (EED-/-) mice were generated by deep sequencing, in triplicate,using Illumina HiSeq 2500 system.All RNA-Seq data were aligned to mouse genome version mm10 using Salmon (v.1.1.0) .We use cluster software and Euclidean distance matrix for the hierarchical clustering analysis of the expressed gene and sample program at the same time, the clustering results can be viewed with javaTreeview. qRT-PCR validation was performed using SYBR Green assays. we performed ChIP-seq using freshly isolated P14 from Wild Type and EED-/- cerebellar tissues . DNA libraries generated from ChIP-DNA and input-DNA were deep-sequenced using HiSeq single-end 50bp. Results:Only transcripts that showed more than 1.5-fold differential expression compared to control were subjected to relevance network analysis. 3149 candidate genes that were differentially expressed with biological functional groups as compared between WT and EED-/- mice .We found that the enrichment of H3K27me3 was decreased H3K27ac was highly increased in EED-/- mice. Conclusions: Our study represents the first detailed analysis of Wild Type and EED-/ mice cerebellum transcriptomes, with three biologic replicates, generated by RNA-seq technology. We report the application of ChIP-sequencing technology for high-throughput profiling of histone modifications in cerebellar cells. Cerebellum mRNA profiles and histone modifications of 14-day-old wild-type (WT) and Embryonic Ectoderm Development conditional knockout (EED-/-) mice研究目的：本研究旨在对比野生型（Wild Type）与EED基因条件性敲除（EED-/-）小鼠小脑的转录组测序（RNA-seq）结果与定量反转录聚合酶链反应（quantitative reverse transcription polymerase chain reaction, qRT-PCR）筛选的差异表达基因，以筛选目标靶基因；同时通过染色质免疫共沉淀测序（chromatin-immunoprecipitation coupled to sequencing, ChIP-seq）技术，解析差异表达基因与H3K27me3、H3K27ac及EED蛋白的结合占据特征之间的关联。 方法部分：本研究采用Illumina HiSeq 2500测序平台，对14日龄（P14）野生型（Wild Type, WT）及胚胎外胚层发育基因条件性敲除（Embryonic Ectoderm Development conditional knockout, EED-/-）小鼠的小脑mRNA进行深度测序，设置3次生物学重复。所有RNA-seq数据通过Salmon（v1.1.0）比对至小鼠基因组mm10版本。同时，本研究使用聚类软件结合欧氏距离矩阵，对表达基因及样本开展分层聚类分析，聚类结果可通过javaTreeview进行查看。采用SYBR Green法完成qRT-PCR验证实验。此外，本研究从野生型与EED-/-小鼠新鲜分离的P14龄小脑组织中获取样本，开展ChIP-seq实验：将ChIP富集的DNA与输入对照DNA构建文库后，使用HiSeq平台进行单端50bp深度测序。 结果部分：本研究仅对相较于对照组表达差异倍数≥1.5的转录本进行关联网络分析，最终在野生型与EED-/-小鼠间筛选得到3149个差异表达候选基因，这些基因富集于不同生物学功能群组。研究发现，在EED-/-小鼠小脑中，H3K27me3的富集水平显著降低，而H3K27ac的富集水平则显著升高。 结论部分：本研究首次通过RNA-seq技术结合3次生物学重复，对野生型与EED-/-小鼠的小脑转录组进行了详细分析；同时验证了ChIP-seq技术在小脑细胞组蛋白修饰高通量谱分析中的应用价值。本研究获取了14日龄野生型（WT）及胚胎外胚层发育基因条件性敲除（EED-/-）小鼠的小脑mRNA表达谱与组蛋白修饰数据。