InDrs. Wessels, Hirsekorn, Ohler and Mukherjee from the Max Delbrück Center for Molecular Medicine published a RIP-Seq protocol in the recent Post-Transcriptional Gene Regulation issue of Methods in Molecular Biology. RIP-Seq is used to uncover genome-wide RNA transcripts that interact with a specific protein or protein complex.
In this assay, ribonucleoprotein (RNP) complexes are immunoprecipitated (RIP) from cell lysates. Associated RNAs are then isolated from these RNP complexes. After the associated RNAs were isolated, these researchers used the NEXTflex™ Rapid Directional qRNA-Seq™ Kit for library preparation. Resulting libraries are suitable for sequencing on any Illumina sequencing platform.
Unlike UV cross-linking and immunoprecipitation (CLIP-Seq) methods, RIP-Seq allows for the detection of RNA components of RNPs that are not directly bound to the RBP of interest. This is particularly relevant when interrogating multicomponent RNPs such as the exon junction complex. RIP-Seq provides whole-transcript-level binding information rather than the site-level resolution of CLIP-Seq methods. Comparisons have shown that RNAs with more CLIP-defined binding sites are more likely to be enriched in the RIP, suggesting that RIP identifies the more stably associated RNAs.
Wessels, H.-H., Hirsekorn, A., Ohler, U. and Mukherjee, N. (2015) Identifying RBP Targets with RIP-seq. Post-Transcriptional Gene Regulation. Methods in Molecular Biology. 1358. 141-152.