27Current methods for the identification of RNA-protein interactions require a quantity and 28 quality of sample that hinders their application, especially for dynamic biological 29 systems or when sample material is limiting. Here, we present a new approach to enrich 30 RNA-Binding Proteins (RBPs): Orthogonal Organic Phase Separation (OOPS), which is 31 compatible with downstream proteomics and RNA sequencing. OOPS enables recovery 32 of RBPs and free protein, or protein-bound RNA and free RNA, from a single sample in 33 an unbiased manner. By applying OOPS to human cell lines, we extract the majority of 34 known RBPs, and importantly identify additional novel RBPs, including those from 35 previously under-represented cellular compartments. The high yield and unbiased 36 nature of OOPS facilitates its application in both dynamic and inaccessible systems. 37 Thus, we have identified changes in RNA-protein interactions in mammalian cells 38 following nocodazole cell-cycle arrest, and defined the first bacterial RNA-interactome. 39 Overall, OOPS provides an easy-to-use and flexible technique that opens new 40 opportunities to characterize RNA-protein interactions and explore their dynamic 41 behaviour. 42 43 44 59 conditions 13 . Moreover, the theoretical dependence on the presence of RNA polyA tails, 60 makes oligo(dT)-based capture methods incompatible for organisms with little or no 61 polyadenylation, such as prokaryotes, or for non-polyadenylated RNAs; thus introducing 62 bias in the RBPs identified. There have been recent attempts to address the oligo(dT) 63 limitations, but these have involved incorporation of modified nucleotides, which by itself 64 restricts its applicability and introduces a bias in transcription-dependent nucleoside-65 incorporation 14-16 . 66 67 2 To address these limitations, we have developed a method based on Acidic 68 Guanidinium Thiocyanate-Phenol-Chloroform (AGPC) phase partition, called 69 Orthogonal Organic Phase Separation (OOPS). AGPC purification permits the recovery 70 of RNA species in an unbiased manner 17,18 . In standard conditions, when lysing cells in 71 AGPC, two distinct phases are formed: RNA migrates to the upper aqueous phase and 72 proteins occupy the lower organic phase. Here, we utilise UV crosslinking at 254 nm to 73 generate RNA-protein adducts that combine the physicochemical properties of both 74 molecules and thus migrate to the aqueous-organic interface 19 . Isolation of the interface 75 allows specific recovery of either RBPs or PBRs by digesting the reciprocal component 76 of the adduct. 77 78 Here, we demonstrate the specificity and versatility of OOPS. Separation of free and 79 protein-bound RNA provides a way to quantify the proportion of RNA crosslinked to 80 protein, enabling precise optimisation of UV dosage. Furthermore, we show that OOPS 81 recovers all crosslinked-RNA (CL-RNA) and thus all crosslinked RBPs. To demonstrate 82 the versatility of OOPS, we analyse RNA-binding changes through cell-cycle 83 progression following noc...