Base-resolution analyses of parent-of-origin and sequence dependent allele specific DNA methylation in the mouse genome (RNA-seq). Mus musculus

Allele specific DNA methylation (ASM) is crucial for genomic imprinting and mammalian development. Here we present a base-resolution, genome-wide allelic DNA methylation map for both CG and non-CG sites in the mouse brain. We found parent-of-origin dependent (imprinted) ASM at 1,952 CGs which form 55 discrete clusters. This uncovers 31 reported differentially methylated regions (DMRs), including virtually all known germline DMRs, and 24 novel candidate DMRs with some occurring at microRNA genes. In the same adult tissue we also report a surprising presence of non-CG methylation with some showing evidence of imprinting. Finally, we identified sequence dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Our genome-wide ASM map should help with understanding the epigenetic differences between two parental genomes in mammals. Overall design: The crosses of the two mouse strains 129x1/SvJ (129) and Cast/EiJ (Cast) were performed at Jackson Laboratories (http://jaxmice.jax.org/) and the male mice F1 offspring and males of each of the two parental strains were shipped to investigator laboratories at 8 to 9 weeks of age. The frontal cortex from the F1 crosses of the two mouse lines was dissected and the RNA was isolated followed by two DNAseI treatments to remove DNA contaminant. The presence of DNA in the RNA was tested using quantitative PCR and probes designed to span exons. The quality of the RNA was determined by the Agilent 2100 Bioanalyzer prior to the construction of the libraries. RNA was treated with RiboMinus (Invitrogen, Carlsbad CA) to remove the ribosomal RNA. The methods for the preparation of the libraries are outlined in the Whole Transcriptome library preparation for SOLiD sequencing protocol (https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_065852.pdf). Briefly, RNA depleted of ribosomal RNA was fragmented using RNAseIII. Purified RNA was hybridized and ligated to primers then converted to cDNA using reverse transcriptase. The cDNA was size selected to contain 50 to 150 bp inserts then purified and amplified prior to sequencing. Libraries were constructed with RNA from three independent mice (3 biological replicates) for each of the experimental crosses. Sequencing was performed at EdgeBio (http://www.edgebio.com/) and the data from the 3 mice for each experimental cross line were combined given the high correlation between each of the libraries (R >0.98).

等位基因特异性DNA甲基化（Allele specific DNA methylation, ASM）对于基因组印记及哺乳动物发育具有关键作用。本研究针对小鼠大脑中的CG位点与非CG位点，构建了碱基分辨率的全基因组ASM图谱。我们在1952个CG位点上发现了亲本来源依赖型（印记型）的ASM，这些位点共同构成55个独立的簇状区域。该图谱共揭示31个已报道的差异甲基化区域（differentially methylated regions, DMRs），其中几乎涵盖所有已知的生殖系DMRs；同时还鉴定出24个全新的候选DMRs，部分候选区域位于微小RNA基因区域内。在同一成年脑组织中，我们还意外发现非CG甲基化的存在，其中部分位点呈现印记特征。最后，我们在131765个CG位点上鉴定出序列依赖性的ASM。值得关注的是，这些位点的甲基化水平显著依赖于其紧邻的侧翼碱基，这使得我们能够明确哺乳动物DNA甲基化机制所具有的保守序列偏好性。本研究构建的全基因组ASM图谱，将为解析哺乳动物双亲基因组间的表观遗传差异提供重要参考。
实验设计：本研究于杰克逊实验室（Jackson Laboratories, http://jaxmice.jax.org/）完成两个小鼠品系129x1/SvJ（简称129）与Cast/EiJ（简称Cast）的杂交实验；将F1代雄性子代及两个亲本品系的雄性小鼠在8~9周龄时运送至研究者实验室。解剖分离两个品系杂交获得的F1代小鼠前额叶皮层，提取总RNA后进行两次DNA酶I处理以去除DNA污染物。通过跨外显子设计的探针结合定量聚合酶链式反应（quantitative PCR, qPCR），检测RNA样品中是否存在DNA残留。在文库构建前，使用安捷伦2100生物分析仪（Agilent 2100 Bioanalyzer）检测RNA样品的质量。使用RiboMinus试剂盒（Invitrogen, Carlsbad CA）处理RNA样品，以去除核糖体RNA。文库制备方法参考《用于SOLiD测序的全转录组文库制备方案》（https://www3.appliedbiosystems.com/cms/groups/mcb_support/documents/generaldocuments/cms_065852.pdf）。简要流程如下：去除核糖体RNA的RNA样品经RNA酶III（RNAseIII）进行片段化；纯化后的RNA与引物杂交并连接，随后通过反转录酶反转录为cDNA；对cDNA进行片段大小筛选，保留插入片段长度为50~150 bp的产物，之后进行纯化与扩增，最终用于测序。每个杂交实验组均采用3只独立小鼠的RNA构建文库，即3个生物学重复。测序工作由EdgeBio公司（http://www.edgebio.com/）完成；由于各文库间相关性极高（相关系数R>0.98），因此将每个杂交实验组的3只小鼠的测序数据进行合并。