Sexton Alec N, Machyna Martin, Simon Matthew D
Department of Molecular Biophysics & Biochemistry, Yale University, 27392, New Haven, CT, 06511, USA.
Chemical Biology Institute, Yale University, West Haven, CT, 06516, USA.
Methods Mol Biol. 2016;1480:87-97. doi: 10.1007/978-1-4939-6380-5_8.
There are numerous recent cases where chromatin modifying complexes associate with long noncoding RNA (lncRNA), stoking interest in lncRNA genomic localization and associated proteins. Capture Hybridization Analysis of RNA Targets (CHART) uses complementary oligonucleotides to purify an RNA with its associated genomic DNA or proteins from formaldehyde cross-linked chromatin. Deep sequencing of the purified DNA fragments gives a comprehensive profile of the potential lncRNA biological targets in vivo. The combined identification of the genomic localization of RNA and its protein partners can directly inform hypotheses about RNA function, including recruitment of chromatin modifying complexes. Here, we provide a detailed protocol on how to design antisense capture oligos and perform CHART in tissue culture cells.
近期有许多染色质修饰复合物与长链非编码RNA（lncRNA）相关联的案例，这引发了人们对lncRNA基因组定位及相关蛋白的兴趣。RNA靶标的捕获杂交分析（CHART）利用互补寡核苷酸从甲醛交联的染色质中纯化与其相关的基因组DNA或蛋白的RNA。对纯化的DNA片段进行深度测序可全面了解体内潜在的lncRNA生物学靶标。RNA及其蛋白伙伴的基因组定位的联合鉴定能够直接为关于RNA功能的假说提供信息，包括染色质修饰复合物的募集。在此，我们提供一份关于如何设计反义捕获寡核苷酸以及在组织培养细胞中进行CHART的详细方案。
