期刊
NATURE PROTOCOLS
卷 15, 期 8, 页码 2568-2588出版社
NATURE RESEARCH
DOI: 10.1038/s41596-020-0344-2
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资金
- Medical Research Council [5TR00]
- Wellcome Trust [110170/Z/15/Z, 110071/Z/15/Z.]
- Biotechnology and Biological Sciences Research Council [BB/M006700/1] Funding Source: researchfish
- Medical Research Council [MC_UP_A600_1023, MC_UU_00025/7] Funding Source: researchfish
- Wellcome Trust [110071/A/15/Z] Funding Source: researchfish
- BBSRC [BB/N010493/1] Funding Source: UKRI
- MRC [MC_UP_A600_1023, MC_UU_00025/7] Funding Source: UKRI
RNA-protein interactions play a pivotal role in cell homeostasis and disease, but current approaches to study them require a considerable amount of starting material, favor the recovery of only a subset of RNA species or are complex and time-consuming. We recently developed orthogonal organic phase separation (OOPS): a quick, efficient and reproducible method to purify cross-linked RNA-protein adducts in an unbiased way. OOPS avoids molecular tagging or the capture of polyadenylated RNA. Instead, it is based on sampling the interface of a standard TRIzol extraction to enrich RNA-binding proteins (RBPs) and their cognate bound RNA. OOPS specificity is achieved by digesting the enriched interfaces with RNases or proteases to release the RBPs or protein-bound RNA, respectively. Here we present a step-by-step protocol to purify protein-RNA adducts, free protein and free RNA from the same sample. We further describe how OOPS can be applied in human cell lines,Arabidopsis thaliana,Schizosaccharomyces pombeandEscherichia coliand how it can be used to study RBP dynamics.
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