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Optimized protocols for RNA-induced silencing complex assembly and cleavage in cultured Drosophila cells

Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lys...

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Detalles Bibliográficos
Autores principales: Gao, Yajie, Zhu, Yuanxiang, Sun, Qinmiao, Chen, Dahua
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9344023/
https://www.ncbi.nlm.nih.gov/pubmed/35928005
http://dx.doi.org/10.1016/j.xpro.2022.101596
Descripción
Sumario:Here, we provide an optimized RNA-induced silencing complex (RISC) assembly and cleavage protocol in vitro without using radiolabeled RNA. The protocol is useful to characterize the biochemical properties of the RISC. We describe the preparation of RNA probes, the target RNA, and Drosophila cell lysates for RISC assembly assay. We then detail AGO1 complexes immunoprecipitation for RISC cleavage assay. This protocol can detect RISC assembly and cleavage products within 5 days. Moreover, it can detect 5′- and 3′-cleavage products simultaneously. For complete details on the use and execution of this protocol, please refer to Gao et al. (2022).