Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins.
Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins.
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DOI：
10.1016/j.xpro.2021.100819
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发表时间：
2021-12-17
期刊：
影响因子：
--
通讯作者：
Lee JT
中科院分区：
文献类型：
--
作者：
Rosenberg M;Levy V;Maier VK;Kesner B;Blum R;Lee JT
在“变性交联RNA免疫沉淀”（dCLIP）方案中分离蛋白质-RNA复合物是基于生物素标记感兴趣的蛋白质、体内UV交联RNA与蛋白质、RNA酶保护测定以及在变性条件下在链霉亲和素柱上分离RNA-蛋白质复合物。由于传统的基于抗体的CLIP测定法应用于Polycomb复合物具有挑战性，因此dCLIP已成功应用并产生小RNA足迹，从中可以进行从头基序分析以鉴定RNA结合基序。有关本方案使用和执行的完整详细信息，请参阅。dCLIP生物标记感兴趣的蛋白质以鉴定体内交联的RNA相互作用物生物素-链霉亲和素纯化系统实现变性洗涤条件dCLIP成功地应用于染色质修饰蛋白质复合物dCLIP允许RNA结合位点的高分辨率作图和从头基序分析在“变性交联RNA免疫沉淀”（dCLIP）方案中蛋白质-RNA复合物的分离基于生物素-链霉亲和素纯化系统。标记感兴趣的蛋白质，体内UV交联RNA与蛋白质，RNA酶保护测定，和在变性条件下在链霉亲和素柱上分离RNA-蛋白质复合物。由于传统的基于抗体的CLIP测定法应用于Polycomb复合物具有挑战性，因此dCLIP已成功应用并产生小RNA足迹，从中可以进行从头基序分析以鉴定RNA结合基序。
The isolation of protein-RNA complexes in the “denaturing cross-linked RNA immunoprecipitation” (dCLIP) protocol is based on biotin-tagging proteins of interest, UV cross-linking RNA to protein in vivo, RNase protection assay, and isolating RNA-protein complexes under denaturing conditions over a streptavidin column. Insofar as conventional antibody-based CLIP assays have been challenging to apply to Polycomb complexes, dCLIP has been applied successfully and yields small RNA footprints from which de novo motif analysis can be performed to identify RNA binding motifs. For complete details on the use and execution of this protocol, please refer to. dCLIP biotags a protein of interest to identify cross-linked RNA interactors in vivo Biotin-streptavidin purification system enables denaturing washing conditions dCLIP is successfully applied to chromatin-modifying protein complexes dCLIP allows high-resolution mapping of RNA binding sites and de novo motif analysis The isolation of protein-RNA complexes in the “denaturing cross-linked RNA Immunoprecipitation” (dCLIP) protocol is based on biotin-tagging proteins of interest, UV cross-linking RNA to protein in vivo, RNase protection assay, and isolating RNA-protein complexes under denaturing conditions over a streptavidin column. Insofar as conventional antibody-based CLIP assays have been challenging to apply to Polycomb complexes, dCLIP has been applied successfully and yields small RNA footprints from which de novo motif analysis can be performed to identify RNA binding motifs.
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影响因子：
16.8
作者：
通讯作者：
--
DOI：
10.1007/978-1-4939-7204-3_14
发表时间：
2017-01-01
期刊：
MRNA PROCESSING: METHODS AND PROTOCOLS
影响因子：
--
作者：
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影响因子：
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