Efficient recovery of the RNA-bound proteome and protein-bound transcriptome using phase separation (OOPS)

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 pombe and Escherichia coli and how it can be used to study RBP dynamics.