We present the experimental protocol and data analysis toolbox for multi-contact 4C (MC-4C), a new proximity ligation method tailored to study the higher-order chromatin contact patterns of selected genomic sites. Conventional chromatin conformation capture (3C) methods fragment proximity ligation products for efficient analysis of pairwise DNA contacts. By contrast, MC-4C is designed to preserve and collect large concatemers of proximity ligated fragments for long-molecule sequencing on an Oxford Nanopore or Pacific Biosciences platform. Each concatemer of proximity ligation products represents a snapshot topology of a different individual allele, revealing its multi-way chromatin interactions. By inverse PCR with primers specific for a fragment of interest (the viewpoint) and DNA size selection, sequencing is selectively targeted to thousands of different complex interactions containing this viewpoint. A tailored statistical analysis toolbox is able to generate background models and three-way interaction profiles from the same dataset. These profiles can be used to distinguish whether contacts between more than two regulatory sequences are mutually exclusive or, conversely, simultaneously occurring at chromatin hubs. The entire procedure can be completed in 2 w, and requires standard molecular biology and data analysis skills and equipment, plus access to a third-generation sequencing platform.

我们提出了多接触 4C (MC-4C) 的实验协议和数据分析工具箱，这是一种新的邻近连接方法，专为研究选定基因组位点的高阶染色质接触模式而设计。传统的染色质构象捕获 (3C) 方法将邻近连接产物片段化，以有效分析成对的 DNA 接触。相比之下，MC-4C 旨在保存和收集邻近连接片段的大串联体，以在 Oxford Nanopore 或 Pacific Biosciences 平台上进行长分子测序。邻近连接产物的每个串联体代表不同单个等位基因的快照拓扑结构，揭示其多向染色质相互作用。通过使用特定于感兴趣片段（视点）的引物的反向 PCR 和 DNA 大小选择，测序选择性地针对包含该观点的数千种不同的复杂相互作用。量身定制的统计分析工具箱能够从同一数据集生成背景模型和三向交互配置文件。这些配置文件可用于区分两个以上调节序列之间的接触是否相互排斥，或者相反，是否同时发生在染色质中心。整个过程可在 2 w 内完成，需要标准的分子生物学和数据分析技能和设备，以及使用第三代测序平台。这些配置文件可用于区分两个以上调节序列之间的接触是否相互排斥，或者相反，是否同时发生在染色质中心。整个过程可在 2 w 内完成，需要标准的分子生物学和数据分析技能和设备，以及使用第三代测序平台。这些配置文件可用于区分两个以上调节序列之间的接触是否相互排斥，或者相反，是否同时发生在染色质中心。整个过程可在 2 w 内完成，需要标准的分子生物学和数据分析技能和设备，以及使用第三代测序平台。