Genome wide maps of Dmc1 in testis of Hop2 null mice.

We report the application of ChIP-seq targeted at the meiosis-specific protein DMC1 to reveal the genome-wide distribution of initiation of meiotic recombination. The mouse model here employed is Hop2-/- because it is unable to repair the DNA double-stranded breaks and therefore the DMC1 signal is more persistent. We also provide the resulting dataset of ChIP-seq targeted at RAD51 which is not meiosis specific but is also targeted at initiation of recombination loci in meiotic tissue. In addition, we report DMC1 ChIP-seq on wild type mouse pup testis. Finally, we present ChIP-seq targeted at H3K4me3 in testis and liver tissues. Lysates from mice testis cells were sonicated, clarified and DNA protein complexes were isolated with anti Dmc1 or anti Rad51 or H3K4Me3 antibodies. DNA purified from proteins and libraries were prepared according to Illumina's instructions accompanying the genomic DNA Sample preparing kit part number 1000181. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenowpolymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (3’ to 5’ exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 18 cycles and library fragments of 150~250 bp were isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. To prepare Micrococcal Nuclease-digested fractions of chromatin DNA, homogenized testis cells were treated with MN and pelleted. The supernatant containing the digested fraction was separated and the DNA was purified from proteins. The library was prepared the same way as described above and all were sequenced on the Genome Analyzer following the manufacturer's protocols.

本研究报道了针对减数分裂特异性蛋白DMC1的染色质免疫共沉淀测序（ChIP-seq，Chromatin Immunoprecipitation Sequencing）技术的应用，以揭示减数分裂重组起始位点的全基因组分布情况。本研究使用的小鼠模型为Hop2基因敲除（Hop2-/-）品系，因其无法修复DNA双链断裂，故而DMC1的信号会更为持久。本研究同时提供了针对RAD51的ChIP-seq数据集：RAD51并非减数分裂特异性蛋白，但同样可结合减数分裂组织中的重组起始位点。此外，本研究还报道了野生型幼鼠睾丸组织的DMC1 ChIP-seq数据。最后，本研究还提供了睾丸与肝脏组织中H3K4me3的ChIP-seq数据。我们对小鼠睾丸细胞的裂解液进行超声破碎、澄清处理，并通过抗Dmc1、抗Rad51或抗H3K4Me3抗体分离DNA-蛋白质复合物。从蛋白质复合物中纯化得到的DNA，按照Illumina配套的基因组DNA样本制备试剂盒（货号1000181）的说明书进行文库构建。简言之，我们采用T4 DNA聚合酶、大肠杆菌DNA聚合酶I大片段（Klenow聚合酶）与T4多核苷酸酶的组合对DNA进行末端修复。将带磷酸化修饰的平末端DNA经3’→5’核酸酶活性缺失的Klenow片段与dATP处理，使其3’端突出单个A碱基，以适配Illumina接头的3’端单个T碱基突出结构，用于后续连接反应。接头连接完成后，使用Illumina通用引物进行18个循环的PCR扩增，再通过琼脂糖凝胶电泳分离150~250 bp的文库片段。纯化后的DNA将被加载至Illumina流动槽以进行簇生成。若要制备微球菌核酸酶（Micrococcal Nuclease, MN）消化的染色质DNA组分，需将匀浆后的睾丸细胞经MN处理后离心收集沉淀，分离得到含消化组分的上清液，再从中纯化蛋白质结合的DNA。该文库的构建流程与前述一致，所有样本均按照厂商说明书在Illumina Genome Analyzer测序平台上完成测序。