Epigenetic control of meiotic recombination in mammals - Equipment Supplement

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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.