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通过旗-生物素串联纯化通过交叉链接和免疫沉淀对蛋白质-RNA相互作用进行转录组-全过程分析

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Genetica

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JoVE Journal Genetica

Transcriptome-Wide Profiling of Protein-RNA Interactions by Cross-Linking and Immunoprecipitation Mediated by FLAG-Biotin Tandem Purification

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通过旗-生物素串联纯化通过交叉链接和免疫沉淀对蛋白质-RNA相互作用进行转录组-全过程分析

DOI:

10.3791/60730-v

•

08:46 min

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May 18, 2020

•

Xianju Bi², Xuechun Zhang, Xiaohua Shen

¹Tsinghua-Peking Center for Life Sciences, School of Medicine and School of Life Sciences,Tsinghua University, ²Department of Molecular Biology,University of Texas Southwestern Medical Center

Capitoli

00:04Introduction
00:41Crosslinking
02:44Immunoprecipitation
05:31Dephosphorylation and PNK Treatment
06:53Results: WDR43 and LIN28 ^FbioCLIP-seq
08:17Conclusion

Summary

Traduzione automatica

在这里，我们提出了一个改进的 CLIP-seq 协议， ^{称为 Fbio}CLIP-seq 与 FLAG 生物素串联纯化，以确定哺乳动物细胞中RNA结合蛋白（RBPs）的RNA靶点。

Tags

Protein-RNA Interactions CLIP-seq FLAG-Biotin Transcriptome-wide Profiling RBPs Mammalian Cells Direct Protein-bound RNAs Isotope-free SDS-PAGE Membrane Transfer Mouse Embryonic Stem Cells UV Cross-linking Cell Lysis DNase I Treatment FLAG Beads FLAG Peptide Elution

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