RIPseq

RIP-seq, short for RNA immunoprecipitation sequencing, is a technique used to identify RNA molecules that bind to a specific RNA-binding protein (RBP). In a typical native RIP-seq workflow, cells or tissues are lysed under non-denaturing conditions, and an antibody against the RBP is used to immunoprecipitate the RBP-RNA complex. The co-purified RNA is then purified, reverse-transcribed, and prepared for high-throughput sequencing. The resulting reads are mapped to a reference transcriptome or genome to determine which RNA species are enriched in the immunoprecipitated sample compared with input or an IgG control, indicating potential targets of the RBP.

A variant of RIP-seq uses crosslinking, such as formaldehyde or UV irradiation, before lysis to stabilize protein–RNA interactions (crosslinked RIP or X-RIP). Crosslinking can capture weaker or transient associations but may introduce biases and complicate interpretation, particularly related to crosslink-induced artifacts.

Data analysis typically involves aligning reads, quantifying RNA species, and performing enrichment analyses to identify RNAs preferentially associated with the RBP. Results are often reported at the transcript level, with considerations for transcript abundance and experimental controls. Validation and follow-up experiments are common to confirm direct binding and to explore functional consequences.

Applications of RIP-seq include identifying targets and networks of RNA-binding proteins, elucidating roles in mRNA stability, localization, and translation, and providing insights into post-transcriptional regulatory mechanisms. Limitations include dependence on antibody quality, potential co-immunoprecipitation of indirect associations, background binding, and the challenge of distinguishing direct from indirect interactions without crosslinking. See also CLIP-seq methods, which rely on crosslinking for higher-resolution mapping of binding sites.