Orthogonal Organic Phase Separation protocol (2018)

ABSTRACT: Identification of RNA–protein interactions currently requires a quantity and quality of sample that precludes their widespread application, especially for dynamic biological systems or precious samples. Here, we present Orthogonal Organic Phase Separation (OOPS), a new approach to enrich RNA Binding Proteins (RBPs) which is compatible with downstream proteomics and RNA sequencing. The flexibility of OOPS enables recovery of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in an unbiased manner. We have applied OOPS to both eukaryotic and previously inaccessible prokaryotic models, demonstrating its wide applicability, efficacy and consistency. We observe that the proteins identified include the majority of previously known RBPs, as well as novel groups of RBPs, including membrane proteins. Furthermore, applying OOPS to a cell-cycle arrest model, we can determine dynamic changes in RNA–protein interaction. Taken together, OOPS opens new model-independent, easy-to-implement opportunities to characterize RNA–protein interactions and their dynamic behaviour.

INSTRUMENT(S): Orbitrap Fusion Lumos, Q Exactive

ORGANISM(S): Homo Sapiens (human) Escherichia Coli

TISSUE(S): Permanent Cell Line Cell

SUBMITTER: Rayner Queiroz  

LAB HEAD: Prof. Kathryn Lilley

PROVIDER: PXD009668 | Pride | 2018-12-17

REPOSITORIES: Pride