期刊
NATURE PROTOCOLS
卷 14, 期 7, 页码 2036-2068出版社
NATURE PUBLISHING GROUP
DOI: 10.1038/s41596-019-0172-4
关键词
-
资金
- NIH [HG004659, HG007005, GM049369, DK098808]
- Strategic Priority Research Program of the Chinese Academy of Sciences [XDA16010113]
- National Science Foundation of China [31872191, 31472263]
- China Scholarship Council [201606275037]
- National Natural Science Foundation of China [91640115]
Chromatin in higher eukaryotic nuclei is extensively bound by various RNA species. We recently developed a method for in situ capture of global RNA interactions with DNA by deep sequencing (GRID-seq) of fixed permeabilized nuclei that allows identification of the entire repertoire of chromatin-associated RNAs in an unbiased manner. The experimental design of GRID-seq is related to those of two recently published strategies (MARGI (mapping RNA-genome interactions) and ChAR-seq (chromatin-associated RNA sequencing)), which also use a bivalent linker to ligate RNA and DNA in proximity. Importantly, however, GRID-seq also implements a combined experimental and computational approach to control nonspecific RNA-DNA interactions that are likely to occur during library construction, which is critical for accurate interpretation of detected RNA-DNA interactions. GRID-seq typically finds both coding and non-coding RNAs (ncRNAs) that interact with tissue-specific promoters and enhancers, especially super-enhancers, from which a global promoter-enhancer connectivity map can be deduced. Here, we provide a detailed protocol for GRID-seq that includes nuclei preparation, chromatin fragmentation, RNA and DNA in situ ligation with a bivalent linker, PCR amplification and high-throughput sequencing. To further enhance the utility of GRID-seq, we include a pipeline for data analysis, called GridTools, into which key steps such as background correction and inference of genomic element proximity are integrated. For researchers experienced in molecular biology with minimal bioinformatics skills, the protocol typically takes 4-5 d from cell fixation to library construction and 2-3 d for data processing.
作者
我是这篇论文的作者
点击您的名字以认领此论文并将其添加到您的个人资料中。
推荐
暂无数据