Base-resolution analyses of parent-of-origin and sequence dependent allele specific DNA methylation in the mouse genome (ChIP-seq and Methyl-seq)

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. 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. MethylC-Seq Five ug of genomic DNA was extracted from the frontal cortex of the F1 crosses or the parental strains, and was spiked in with 25 ng unmethylated cl857 Sam7 Lambda DNA (Promega, Madison, WI). The DNA was fragmented by sonication to 100-600 bp. Purified DNA fragments were end-repaired and ligated to paired-end cytosine- methylated adapters provided by Illumina. Size-selected adapter-ligated DNA was treated with sodium bisulfite using the EZ DNA methylation-Gold Kit (Zymo Research). The resulting DNA molecules were enriched by PCR, purified and sequenced following standard protocols from Illumina. ChIP-Seq 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. Frozen mouse frontal cortex from the F1 crosses was thawed on ice and processed with a razor blade into small pieces. Tissue was then crosslinked with formaldehyde, washed, homogenized, and proceeded for ChIP. To isolate chromatin, formaldehyde-cross-linked nuclei were sonicated using a Branson 450 Sonifier (Branson, Danbury, CT). ChIP DNA was subjected to end repair, A-tailing, adaptor ligation, and gel purification. Following PCR amplification for 18 cycles, the final libraries were subjected to sequencing.

等位基因特异性DNA甲基化（Allele Specific DNA Methylation，ASM）对基因组印记和哺乳动物发育至关重要。本研究提供了小鼠大脑中CG和非CG位点的单碱基分辨率全基因组等位基因DNA甲基化图谱。我们在1952个CG位点上发现了亲本起源依赖性（印记型）ASM，这些位点形成55个独立簇。本次研究共鉴定出31个已报道的差异甲基化区域（Differentially Methylated Regions，DMRs），几乎涵盖所有已知的生殖系DMRs，以及24个新的候选DMRs，其中部分位于微小RNA（microRNA）基因区域。在同一成年脑组织中，我们还意外发现了非CG甲基化现象，其中部分位点表现出印记特征。最后，我们在131765个CG位点上鉴定出序列依赖性ASM。有趣的是，这些位点的甲基化水平强烈依赖于紧邻的碱基，这使得我们能够明确哺乳动物DNA甲基化酶的保守序列偏好性。我们的全基因组ASM图谱将有助于解析哺乳动物双亲基因组之间的表观遗传差异。

本研究使用的两种小鼠品系129x1/SvJ（简称129）和Cast/EiJ（简称Cast）的杂交实验在杰克逊实验室（Jackson Laboratories，http://jaxmice.jax.org/）完成，F1代雄性子代以及两个亲本品系的雄性小鼠在8至9周龄时被运送至研究者实验室。

MethylC-Seq实验流程：从F1杂交子代或亲本品系的前额叶皮层中提取5μg基因组DNA，并掺入25ng未甲基化的cl857 Sam7 Lambda DNA（Promega，麦迪逊，威斯康星州）作为内参。通过超声破碎将DNA片段化为100-600bp。将纯化的DNA片段进行末端修复，并连接Illumina提供的双端胞嘧啶甲基化接头。使用EZ DNA甲基化黄金试剂盒（Zymo Research）对经过大小选择的接头连接DNA进行亚硫酸氢盐处理。按照Illumina标准流程对所得DNA分子进行PCR富集、纯化后测序。

染色质免疫共沉淀测序（ChIP-Seq）实验流程：两种小鼠品系129x1/SvJ（129）和Cast/EiJ（Cast）的杂交实验同样在杰克逊实验室（http://jaxmice.jax.org/）完成，F1代雄性子代以及两个亲本品系的雄性小鼠在8至9周龄时被运送至研究者实验室。将F1杂交子代的冰冻小鼠前额叶皮层在冰上解冻，用剃须刀片切成小块。随后将组织用甲醛交联、洗涤、匀浆，进行ChIP实验。为分离染色质，使用Branson 450超声破碎仪（Branson，丹伯里，康涅狄格州）对甲醛交联的细胞核进行超声破碎。将ChIP得到的DNA进行末端修复、加A尾、接头连接和凝胶纯化。经过18个循环的PCR扩增后，对最终的文库进行测序。