Lambing Christophe, Choi Kyuha, Blackwell Alexander R, Henderson Ian R
Department of Plant Sciences, University of Cambridge, Cambridge, UK.
Department of Life Sciences, Pohang University of Science and Technology, Pohang, Gyeongbuk, Republic of Korea.
Methods Mol Biol. 2020;2061:219-236. doi: 10.1007/978-1-4939-9818-0_16.
During meiosis recombination occurs between homologous chromosomes which can result in reciprocal exchanges of genetic information, called crossovers. Crossover rate is heterogeneous within the genome, with local regions having a significantly higher recombination rate relative to the genome average. These regions are termed hotspots and typically occur with widths of kilobases. Therefore, there is a need to profile recombination factors at a similar resolution during meiosis via techniques such as chromatin immunoprecipitation (ChIP). Here we describe a ChIP protocol, combined with high throughput sequencing (ChIP-seq) optimised for analysis of meiotically expressed proteins in Arabidopsis thaliana flowers. We provide methods to (1) isolate nuclei and prepare the chromatin for shearing, (2) immunoprecipitate DNA molecules cross-linked to a protein of interest, (3) to size-select and purify immunoprecipitated DNA molecules, and (4) to prepare DNA sequencing libraries suitable for high-throughput sequencing. Together, these methods allow the detection of binding sites for meiotic proteins in the Arabidopsis genome at high resolution, which will provide insights into relationships between meiotic chromosome organization, chromatin and recombination.
在减数分裂过程中，同源染色体之间会发生重组，这可能导致遗传信息的相互交换，即交叉互换。交叉互换率在基因组内是不均匀的，相对于基因组平均水平，局部区域的重组率显著更高。这些区域被称为热点，通常宽度为几千个碱基对。因此，有必要通过染色质免疫沉淀（ChIP）等技术，在减数分裂过程中以类似的分辨率分析重组因子。在这里，我们描述了一种ChIP方案，并结合了高通量测序（ChIP-seq），该方案针对拟南芥花中减数分裂表达的蛋白质分析进行了优化。我们提供了以下方法：（1）分离细胞核并制备用于剪切的染色质，（2）免疫沉淀与感兴趣蛋白质交联的DNA分子，（3）对免疫沉淀的DNA分子进行大小选择和纯化，以及（4）制备适合高通量测序的DNA测序文库。这些方法共同作用，能够在高分辨率下检测拟南芥基因组中减数分裂蛋白的结合位点，这将为深入了解减数分裂染色体组织、染色质和重组之间的关系提供线索。
