Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell.
Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell.
复制标题
将荧光成像与 Hi-C 相结合，研究同一单细胞的 3D 基因组结构。
DOI：
10.1038/nprot.2018.017
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发表时间：
2018
期刊：
影响因子：
14.8
通讯作者：
Lando D
中科院分区：
文献类型：
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作者：
Lando D
荧光成像和Hi-C等染色体构象捕获分析是研究基因组组织的关键工具。然而，传统上，它们是独立进行的，使得这两种类型的数据的集成难以执行。通过将单个细胞核捕获在具有琼脂糖垫的384孔玻璃底板的孔内，我们已经建立了一种允许在同一单个细胞上进行荧光成像和Hi-C处理的方案。该方案与荧光图像并行识别每个单单倍体基因组30，000 - 100，000个染色体接触。接触可以用来计算完整的基因组结构，分辨率超过100 kb，然后可以直接与图像进行比较。使用该方案制备20个单细胞Hi-C文库需要具有分子生物学技术经验的研究人员进行5 d的工作。图像采集和分析需要对荧光显微镜有基本的了解，并且需要一些生物信息学知识来运行这里描述的序列处理工具。
Fluorescence imaging and chromosome conformation capture assays such as Hi-C are key tools for studying genome organization. However, traditionally, they have been carried out independently, making integration of the two types of data difficult to perform. By trapping individual cell nuclei inside a well of a 384-well glass-bottom plate with an agarose pad, we have established a protocol that allows both fluorescence imaging and Hi-C processing to be carried out on the same single cell. The protocol identifies 30,000–100,000 chromosome contacts per single haploid genome in parallel with fluorescence images. Contacts can be used to calculate intact genome structures to better than 100-kb resolution, which can then be directly compared with the images. Preparation of 20 single-cell Hi-C libraries using this protocol takes 5 d of bench work by researchers experienced in molecular biology techniques. Image acquisition and analysis require basic understanding of fluorescence microscopy, and some bioinformatics knowledge is required to run the sequence-processing tools described here.