Next-generation sequencing Facilitates Quantitative Analysis of Wild Type and Arid1a haploinsufficiency mice excitatory neuron Transcriptomes and Genome-wide maps of chromatin state in Wild Type and Ar id1a Haploinsufficiency mice in excitatory neuron

Purpose: The goals of this study are to compare WT and Arid1a in excitatory neuron 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 H3K27ac and Arid1a. Methods: Excitatory neurons mRNA profiles of adult wild-type (WT) and Arid1a Het mice were generated by deep sequencing, in duplicate,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. 3815 candidate genes that were differentially expressed with biological functional groups as compared between WT and Arid1a Het mice .We found that the enrichment of H3K27acwas decreased in Arid1a Het mice. Conclusions: Our study represents the first detailed analysis of Wild Type and Arid1a haploinsufficiency mice excitatory neuron transcriptomes, with two biologic replicates, generated by RNA-seq technology. We report the application of ChIP-sequencing technology for high-throughput profiling of histone modifications in excitatory neurons. mRNA profiles and histone modifications of adult wild-type (WT) and ARID1A haploinsufficiency mice

研究目的：本研究旨在通过RNA测序（RNA-seq）对兴奋性神经元转录组进行分析，并与定量逆转录聚合酶链反应（qRT-PCR）检测的差异表达基因进行对比，以筛选目标基因。同时，我们采用染色质免疫共沉淀联合测序（ChIP-seq）技术，解析差异表达基因与H3K27ac及Arid1a的结合富集关系。
研究方法：本研究利用Illumina HiSeq 2500测序平台，对成年野生型（WT）及Arid1a杂合（Het）小鼠的兴奋性神经元mRNA进行双重复深度测序，获取转录组数据。所有RNA-seq数据均通过Salmon（v.1.1.0）比对至小鼠基因组mm10版本。同时，我们采用聚类软件结合欧氏距离矩阵，对表达基因及样本进行层次聚类分析，聚类结果可通过javaTreeview查看。定量逆转录聚合酶链反应（qRT-PCR）验证实验采用SYBR Green法完成。此外，我们使用新鲜分离的野生型及EED纯合敲除（EED-/-）小鼠P14龄小脑组织进行ChIP-seq实验，由ChIP富集DNA及输入DNA构建的文库均采用HiSeq单端50bp模式进行深度测序。
研究结果：仅将与对照组相比表达差异倍数达1.5倍以上的转录本进行关联网络分析。本研究在WT与Arid1a杂合小鼠之间筛选得到3815个差异表达候选基因，并对其进行了生物学功能基团注释。我们发现，Arid1a杂合小鼠体内H3K27ac的富集水平显著降低。
研究结论：本研究首次通过RNA测序技术，对野生型及Arid1a单倍体剂量不足小鼠的兴奋性神经元转录组进行了详细分析，且实验设置了两次生物学重复。本研究还展示了ChIP测序技术在兴奋性神经元组蛋白修饰高通量分析中的应用，并获取了成年野生型及ARID1A单倍体剂量不足小鼠的mRNA表达谱与组蛋白修饰数据。