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

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

DOI:
10.3791/60730-v

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08:46 min

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May 18, 2020

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, ,
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

Chapters

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

Summary

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Here we present a modified CLIP-seq protocol called FbioCLIP-seq with FLAG-biotin tandem purification to determine the RNA targets of RNA-binding proteins (RBPs) in mammalian cells.

Tags

Keywords: Protein-RNA InteractionsCLIP-seqFLAG-BiotinTranscriptome-wide ProfilingRBPsMammalian CellsDirect Protein-bound RNAsIsotope-freeSDS-PAGEMembrane TransferMouse Embryonic Stem CellsUV Cross-linkingCell LysisDNase I TreatmentFLAG BeadsFLAG Peptide Elution

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