Institute of Pathology, University Medical Center Göttingen, Georg-August University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany.
Institute of Pathology, University Medical Center Göttingen, Georg-August University of Göttingen, Robert-Koch-Str. 40, 37075 Göttingen, Germany; Center for Molecular Medicine Cologne, University and University Hospital of Cologne, Robert-Koch-Str. 21, 50931 Cologne, Germany.
Methods. 2020 Jan 1;170:33-37. doi: 10.1016/j.ymeth.2019.07.003. Epub 2019 Jul 5.
Genome organization is now understood to be tightly linked to all genomic functions. Thus, the high-resolution mapping of higher-order chromosomal structures via 3C-based approaches has become an integral tool for studying transcriptional and cell cycle regulation, signaling effects or disease onset. Nonetheless, 3C-based protocols are not without caveats, like dependencies on fixation conditions, restriction enzyme pervasiveness in crosslinked chromatin and ligation efficiency. To address some of these caveats, we describe here the streamlined iHi-C 2.0 protocol that allows for the genome-wide interrogation of native spatial chromatin contacts without a need for chemical fixation. This approach improves ligation efficiency and presents minimal material losses, and is thus suitable for analysing samples with limiting cell numbers. Following high throughput sequencing, iHi-C 2.0 generates high signal-to-noise and focal maps of the interactions within and between mammalian chromosomes under native conditions.
基因组组织现在被认为与所有基因组功能紧密相关。因此，通过基于 3C 的方法对高级染色体结构进行高分辨率作图已成为研究转录和细胞周期调控、信号效应或疾病发生的重要工具。尽管如此，基于 3C 的方案并非没有缺点，例如对固定条件的依赖性、交联染色质中限制酶的普遍性以及连接效率。为了解决其中的一些缺点，我们在这里描述了简化的 iHi-C 2.0 方案，该方案允许在无需化学固定的情况下对天然空间染色质接触进行全基因组研究。该方法提高了连接效率，并且材料损失最小，因此适用于分析细胞数量有限的样本。在高通量测序之后，iHi-C 2.0 在天然条件下生成哺乳动物染色体内部和之间相互作用的高信噪和焦点图谱。
