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Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)
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Genetik
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JoVE Journal Genetik
Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

DOI:
10.3791/59913-v

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09:26 min

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July 10, 2019

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, ,
1Department of Molecular Genetics, Center for RNA Biology,The Ohio State University

Bölümler

  • 00:04Başlık
  • 01:40Cell Harvesting and Formaldehyde Treatment
  • 02:50FLAG Immunoprecipitation and RNase I Digestion
  • 04:09Affinity Elution and Magnetic Bead-antibody Conjugation
  • 05:14Second Immunoprecipitation
  • 06:12RNA Extraction and Analysis
  • 07:51Results: RNA and Protein Yields
  • 08:38Conclusion

Özet

Otomatik Çeviri

Otomatik Çeviri

Here, we present a protocol to enrich endogenous RNA binding sites or "footprints" of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

Etiketler

RNA ImmunoprecipitationRIPiT-SeqRNA-protein ComplexesEpitope-tagged ProteinCRISPRImmunoprecipitationRNA ExtractionHEK293 CellsRNA Binding ProteinsRNA ProcessingDiseasesNeuropathiesCancer

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