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Multiplexed transcriptome discovery of RNA-binding protein binding sites by antibody-barcode eCLIP

Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the abi...

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Detalles Bibliográficos
Autores principales: Lorenz, Daniel A., Her, Hsuan-Lin, Shen, Kylie A., Rothamel, Katie, Hutt, Kasey R., Nojadera, Allan C., Bruns, Stephanie C., Manakov, Sergei A., Yee, Brian A., Chapman, Karen B., Yeo, Gene W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group US 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9834051/
https://www.ncbi.nlm.nih.gov/pubmed/36550273
http://dx.doi.org/10.1038/s41592-022-01708-8
Descripción
Sumario:Ultraviolet crosslinking and immunoprecipitation (CLIP) methodologies enable the identification of RNA binding sites of RNA-binding proteins (RBPs). Despite improvements in the library preparation of RNA fragments, the enhanced CLIP (eCLIP) protocol requires 4 days of hands-on time and lacks the ability to process several RBPs in parallel. We present a new method termed antibody-barcode eCLIP that utilizes DNA-barcoded antibodies and proximity ligation of the DNA oligonucleotides to RBP-protected RNA fragments to interrogate several RBPs simultaneously. We observe performance comparable with that of eCLIP with the advantage of dramatically increased scaling while maintaining the same material requirement of a single eCLIP experiment.