哺乳類細胞におけるRIP-seqおよびRIP-qPCRを用いたRNA-タンパク質複合体の評価プロトコル
Jackson B Trotman1, Shuang Li1, Quinn E Eberhard2
1Department of Pharmacology, RNA Discovery Center, and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Abstract:
RNA-protein interactions drive gene regulation, subcellular organization, and noncoding RNA function. Here, we present a protocol for measuring RNA-protein associations in formaldehyde-crosslinked mammalian cells using RNA immunoprecipitation followed by sequencing (RIP-seq) and quantitative PCR (RIP-qPCR). We include steps and best practices for qualifying reagents, preparing cells, and processing and analyzing data, including a standardized set of steps to quantify signal over noise. This protocol is broadly applicable for the study of RNA-protein interactions in cells. For complete details on the use and execution of this protocol, please refer to Trotman et al.1.
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