Li Xingwang, Luo Oscar Junhong, Wang Ping, Zheng Meizhen, Wang Danjuan, Piecuch Emaly, Zhu Jacqueline Jufen, Tian Simon Zhongyuan, Tang Zhonghui, Li Guoliang, Ruan Yijun
The Jackson Laboratory for Genomic Medicine, Farmington, Connecticut, USA.
National Key Laboratory of Crop Genetic Improvement, College of Life Sciences &Technology, Huazhong Agricultural University, Wuhan, China.
Nat Protoc. 2017 May;12(5):899-915. doi: 10.1038/nprot.2017.012. Epub 2017 Mar 30.
Chromatin interaction analysis by paired-end tag sequencing (ChIA-PET) is a robust method for capturing genome-wide chromatin interactions. Unlike other 3C-based methods, it includes a chromatin immunoprecipitation (ChIP) step that enriches for interactions mediated by specific target proteins. This unique feature allows ChIA-PET to provide the functional specificity and higher resolution needed to detect chromatin interactions, which chromosome conformation capture (3C)/Hi-C approaches have not achieved. The original ChIA-PET protocol generates short paired-end tags (2 × 20 base pairs (bp)) to detect two genomic loci that are far apart on linear chromosomes but are in spatial proximity in the folded genome. We have improved the original approach by developing long-read ChIA-PET, in which the length of the paired-end tags is increased (up to 2 × 250 bp). The longer PET reads not only improve the tag-mapping efficiency but also increase the probability of covering phased single-nucleotide polymorphisms (SNPs), which allows haplotype-specific chromatin interactions to be identified. Here, we provide the detailed protocol for long-read ChIA-PET that includes cell fixation and lysis, chromatin fragmentation by sonication, ChIP, proximity ligation with a bridge linker, Tn5 tagmentation, PCR amplification and high-throughput sequencing. For a well-trained molecular biologist, it typically takes 6 d from cell harvesting to the completion of library construction, up to a further 36 h for DNA sequencing and <20 h for processing of raw sequencing reads.
通过双末端标签测序（ChIA-PET）进行染色质相互作用分析是一种用于捕获全基因组染色质相互作用的强大方法。与其他基于3C的方法不同，它包括一个染色质免疫沉淀（ChIP）步骤，该步骤可富集由特定靶蛋白介导的相互作用。这一独特特性使ChIA-PET能够提供检测染色质相互作用所需的功能特异性和更高分辨率，而这是染色体构象捕获（3C）/Hi-C方法所无法实现的。原始的ChIA-PET方案生成短双末端标签（2×20碱基对（bp）），以检测线性染色体上相距较远但在折叠基因组中空间上接近的两个基因组位点。我们通过开发长读长ChIA-PET改进了原始方法，其中双末端标签的长度增加（最长可达2×250 bp）。更长的PET读长不仅提高了标签映射效率，还增加了覆盖相位单核苷酸多态性（SNP）的概率，从而能够识别单倍型特异性染色质相互作用。在这里，我们提供了长读长ChIA-PET的详细方案，包括细胞固定和裂解、超声破碎染色质、ChIP、用桥连接头进行邻近连接、Tn5转座标签化、PCR扩增和高通量测序。对于训练有素的分子生物学家来说，从细胞收获到文库构建完成通常需要6天，DNA测序最多还需要36小时，原始测序读段的处理需要不到20小时。
