Epigenetic control of meiotic recombination in mammals - Equipment Supplement

摘要 母公司项目总结：该设备申请的母公司赠款(R01GM125803)的名称为 哺乳动物减数分裂重组的控制“。该奖项涵盖了实验室的主要研究重点。 分析染色质控制DNA修复与染色体机制之间的相互作用 小鼠减数分裂过程中的运动。我们很大比例的研究生产力是通过产生 野生型和生殖细胞中我们感兴趣的蛋白质染色质结合部位的全基因组图谱 转基因小鼠。这包括精母细胞的纯化，以及随后的染色质免疫沉淀。 通过深层测序(CHIP-SEQ)和RNA-SEQ。这与组织学和细胞学观察是相辅相成的。 睾丸特异基因敲除小鼠精母细胞染色质重构体的生物显微镜分析。 利用芯片序列技术获得并分析全基因组蛋白质染色质结合位点图谱。新一代 ChIP-SEQ图谱：关于鉴定蛋白质结合位点所需的DNA材料的产生，我们 经常使用不同阶段的小鼠精母细胞的浓缩部分来获得交联型DNA 片段-蛋白质复合体，允许我们(在免疫沉淀和DNA测序后)评估 目的蛋白的精确定位及其在特定小鼠基因中的分布变化 背景和生化操作。这种方法在研究染色质方面非常有用。 重塑复合体的组成和亚基功能的特异性，这是对 染色质重构体对染色质构象和DNA修复的影响。 目前所需的仪器和设备。由于设备有限，我们的实验室面临着技术挑战 用于制备DNA-蛋白质的现有仪器的可及性和质量(详述如下) DNA深度测序前的加合物和DNA文库的制备。 ChIP-SEQ处理和所需仪器：在获得小鼠精母细胞的浓缩部分后， DNA-蛋白质复合体是用多聚甲醛固定的，DNA必须被精确粉碎，在特定的 我们之前使用聚焦超声仪确定的DNA范围大小(请求Covaris E220进化)。 在程序的这个阶段，仔细测量DNA的大小和质量以评估效率至关重要 并允许产生用于测序的合适的DNA文库。量与质 测量需要使用至少两种替代测量方法。正是在这里，微体积UV-Vis 小体积的分光光度计和荧光计测量(请求Qubit Flex)一起允许您 获取有关DNA样本浓度和质量的最完整信息。这是必不可少的 防止下游成本高昂的故障排除。在下一步，DNA-蛋白质样本进行免疫沉淀。 有了特定的抗体和蛋白质，DNA的交联性就会逆转。在这里，DNA大小需要被精确评估 (请求Agilent 4510 Tapestation)，并再次对样品进行量化。

Summary Summary of parent project: The parent grant (R01GM125803) for this equipment request is entitled “Epigenetic Control of Meiotic Recombination in Mammals”. This award covers the primary research focus of the laboratory analyzing the interplay between chromatin-based control of DNA repair and the mechanics of chromosome movement in mouse meiosis. A large percentage of our research productivity is achieved through generation of genome wide maps of chromatin binding sites for our proteins of interest in wild type and germ cell genetically engineered mice. This includes, purification of spermatocyte cells, and chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and RNA-seq. This is complemented with observation of histological and cell biology microscopic analysis of testis specific knockout mice for chromatin remodelers in mice spermatocytes. Obtaining and analyzing genome wide profiles of protein chromatin binding sites using ChIP-seq. Generation of ChIP-seq profiles: Regarding generation of DNA material necessary for identification of protein binding sites, we often employ enriched fractions of mouse spermatocytes at different stages to obtain crosslinked DNA fragments-protein complexes that allow us (after immunoprecipitation and DNA sequencing) assessing the precise localization of protein of interest and how their distribution changes in specific mouse genetic backgrounds and biochemical manipulations. This approach has shown to be very useful in studying chromatin remodeling complexes composition and specificity of subunit functions, which are direct measurements of chromatin remodeler activity on chromatin conformation and DNA repair. Current instrumentation and equipment needed. Our laboratory confronts technical challenges due to the limited accessibility and quality of the existent instrumentation (detailed below) employed to prepare DNA-protein adducts and preparation of DNA libraries previous to DNA deep sequencing. ChIP-seq processing and required instrumentation: After enriched fraction of mouse spermatocytes are obtained, DNA-protein complexes are fixed with paraformaldehyde and DNA must be precisely shredded, in a specific DNA range size that we previously determined, using a focused ultrasonicator (request Covaris E220 evolution). At this stage of the procedure, is critical that DNA size and quality is carefully measured to evaluate the efficiency of previous steps and allow the generation of suitable DNA libraries for sequencing. Quantity and quality measurement require the use of at least two alternative measures. It is here that Microvolume UV-Vis Spectrophotometer and Fluorometer measurement (request Qubit flex) of small volumes together allow you to obtain the most complete information about the concentration and quality DNA samples. This is essential to prevent costly troubleshooting downstream. In the next step, DNA-protein samples undergo immunoprecipitation with specific antibodies and protein-DNA crosslink is reversed. Here, DNA size needs to be precisely assessed (request Agilent 4510 Tapestation), and samples are quantified again.