Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.
Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, the Netherlands.
Methods. 2020 Jan 1;170:17-32. doi: 10.1016/j.ymeth.2019.07.014. Epub 2019 Jul 26.
Chromosome conformation capture (3C) methods measure DNA contact frequencies based on nuclear proximity ligation, to uncover in vivo genomic folding patterns. 4C-seq is a derivative 3C method, designed to search the genome for sequences contacting a selected genomic site of interest. 4C-seq employs inverse PCR and next generation sequencing to amplify, identify and quantify its proximity ligated DNA fragments. It generates high-resolution contact profiles for selected genomic sites based on limited amounts of sequencing reads. 4C-seq can be used to study multiple aspects of genome organization. It primarily serves to identify specific long-range DNA contacts between individual regulatory DNA modules, forming for example regulatory chromatin loops between enhancers and promoters, or architectural chromatin loops between cohesin- and CTCF- associated domain boundaries. Additionally, 4C-seq contact profiles can reveal the contours of contact domains and can identify the structural domains that co-occupy the same nuclear compartment. Here, we present an improved step-by-step protocol for sample preparation and the generation of 4C-seq sequencing libraries, including an optimized PCR and 4C template purification strategy. In addition, a data processing pipeline is provided which processes multiplexed 4C-seq reads directly from FASTQ files and generates files compatible with standard genome browsers for visualization and further statistical analysis of the data such as peak calling using peakC. The protocols and the pipeline presented should readily allow anyone to generate, visualize and interpret their own high resolution 4C contact datasets.
染色体构象捕获（3C）方法基于核邻近连接测量 DNA 接触频率，以揭示体内基因组折叠模式。4C-seq 是一种衍生的 3C 方法，旨在搜索基因组中与所选基因组感兴趣区域接触的序列。4C-seq 采用反向 PCR 和下一代测序来扩增、识别和定量其邻近连接的 DNA 片段。它基于有限数量的测序读数为选定的基因组区域生成高分辨率的接触谱。4C-seq 可用于研究基因组组织的多个方面。它主要用于识别单个调节 DNA 模块之间的特定长距离 DNA 接触，例如在增强子和启动子之间形成调节染色质环，或在带有黏合蛋白和 CTCF 相关域边界的结构染色质环之间。此外，4C-seq 接触谱可以揭示接触域的轮廓，并可以识别共同占据同一核区室的结构域。在这里，我们提出了一种改进的分步协议，用于样品制备和生成 4C-seq 测序文库，包括优化的 PCR 和 4C 模板纯化策略。此外，还提供了一个数据处理管道，该管道可直接从 FASTQ 文件处理多路复用 4C-seq 读取，并生成与标准基因组浏览器兼容的文件，用于可视化和进一步分析数据，例如使用 peakC 进行峰调用。所提出的方案和管道应易于任何人生成、可视化和解释他们自己的高分辨率 4C 接触数据集。
