RIPsequencing

RIP sequencing, or RIP-seq, is a technique used to identify RNA molecules that are bound by a specific RNA-binding protein (RBP) within cells. It combines immunoprecipitation of RBP–RNA complexes with high-throughput sequencing to reveal the RNA targets of the protein.

In typical workflows, cells or tissues are lysed under conditions that preserve RBP–RNA interactions. An antibody against the RBP is used to immunoprecipitate the complexes, which are then processed to recover the associated RNAs. The RNA is converted to cDNA and sequenced. Sequencing reads are aligned to a reference genome or transcriptome, and enriched RNAs are identified by comparing the immunoprecipitation sample to an input or control. Some protocols include crosslinking prior to lysis to stabilize interactions, while native RIP omits crosslinking.

Data analysis produces a list of RNA targets of the RBP, and, depending on design and depth, may provide gene-level targets or binding-site information along transcripts. Results are often integrated with expression data or other genomic datasets to infer regulatory roles and networks.

Applications of RIP-seq include mapping post-transcriptional regulatory networks, identifying mRNA targets of splicing factors or RNA transport proteins, and studying impacts on mRNA stability, localization, and translation. The method complements other approaches that study RNA–protein interactions by providing a relatively straightforward means to profile target RNAs.

Limitations include dependence on antibody quality and specificity, potential recovery of non-specific or indirect RNAs, and generally lower resolution of binding sites compared with CLIP-based methods. RIP-seq is commonly used alongside higher-resolution techniques for comprehensive mapping of RNA–protein interactions.