Epigenetic control of meiotic recombination in mammals - Equipment Supplement

总结 母项目概要：本设备申请的母资助（R 01 GM 125803）标题为“表观遗传学 哺乳动物减数分裂的控制”。该奖项涵盖了实验室的主要研究重点 分析基于染色质的DNA修复控制与染色体力学之间的相互作用 小鼠减数分裂中的运动。我们的研究生产力的很大一部分是通过生成 我们感兴趣的蛋白质在野生型和生殖细胞遗传学上的染色质结合位点的全基因组图谱 基因改造小鼠这包括精母细胞的纯化和染色质免疫沉淀 通过深度测序（ChIP-seq）和RNA-seq。这与组织学和细胞学观察相补充。 睾丸特异性基因敲除小鼠精母细胞染色质重塑的生物显微镜分析。 使用ChIP-seq获得和分析蛋白质染色质结合位点的全基因组谱。一代 ChIP-seq图谱：关于鉴定蛋白质结合位点所需的DNA材料的生成，我们 通常使用不同阶段的小鼠精母细胞的富集级分来获得交联DNA 片段-蛋白质复合物，使我们（免疫沉淀和DNA测序后）评估 目的蛋白的精确定位以及它们在特定小鼠遗传学中的分布如何变化 背景和生化操作。这种方法在研究染色质方面非常有用 重塑复合物的组成和亚基功能的特异性，这是直接测量 染色质重构活性对染色质构象和DNA修复的影响。 目前所需的仪器和设备。我们的实验室面临着技术挑战，由于有限的 用于制备DNA-蛋白质的现有仪器的可及性和质量（详述如下） 加合物和DNA文库的制备，然后进行DNA深度测序。 ChIP-seq处理和所需仪器：在获得小鼠精母细胞的富集级分后， DNA-蛋白质复合物用多聚甲醛固定，DNA必须精确地粉碎，在特定的条件下， 我们之前使用聚焦超声仪确定的DNA范围大小（要求Covaris E220 evolution）。 在程序的这个阶段，仔细测量DNA的大小和质量以评估效率是至关重要的 并且允许产生用于测序的合适的DNA文库。数量和质量 测量需要使用至少两种替代测量。正是在这里， 小体积的分光光度计和荧光计测量（请求Qubit flex）一起允许您 获得有关DNA样品浓度和质量的最完整信息。这对于以下方面至关重要： 避免下游昂贵的故障排除。下一步，DNA-蛋白质样品进行免疫沉淀 与特异性抗体和蛋白质-DNA交联被逆转。在这里，需要精确评估DNA大小 （要求Agilent 4510 Tapestation），并再次定量样品。

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.