CLIP-seq for RNA-Protein Interactions Analysis

RNA binding protein (RBPs) plays an important role in post-transcriptional regulation, such as the regulation of mRNA splicing, translation and degradation in different biological environments. The recognition and characterization of RNA substrate is the key step to decipher the function and molecular mechanism of the target RBPs.

The high-throughput sequencing of RNA fragments isolated by crosslinking immunoprecipitation (CLIP-seq) is one of the standard techniques for the identification of wide binding sites in the transcriptome of target RNA binding proteins. Using this technique, it is possible to label the natural binding site of RBPs, and may allow the identification of the entire target library of RBPs, thereby contributing to a better understanding of post-transcriptional regulatory networks and mechanisms.

Introduction of CLIP-seq

CLIP-seq, also known as HITS-CLIP, combines UV cross-linking and immunoprecipitation with high-throughput sequencing to identify the binding site of RBPs. CLIP-seq depends on the crosslinking induced mutation site (CIMS) and the local protein-RNA binding site. Because CIMS is reproducible, a higher sequencing depth can distinguish CIMS from technical errors.

The workflow of CLIP-seq is as follows:

• CLIP-seq starts with in vivo cross-linking of RNA protein complexes using ultraviolet light. Cell lysis and immunoprecipitation are used to separate proteins of interest;
• Rinse to remove free RNA and attach RNA adaptors at the 3' end;
• Protease K digestion. This will leave a peptide at the cross-link site to alter the chemical structure of the nucleotide;
• The 5' RNA adaptor is connected to synthesize cDNA by reverse transcription;
• PCR and sequencing of cDNA.

Fig.1 Workflow of CLIP-seq.

Advantages of Our CLIP-seq Technology

• Crosslinking stabilizes the protein-target binding: the interaction between RNA and protein identified by clip reflects the in vivo interaction caused by the nature of UV crosslinking;
• Highly accurate mapping of RNA-protein interactions: CLIP can recognize RNA fragments that bind directly to the target RBP, but not to other proteins that interact with the target RBP;
• To map the RNA-protein binding sites on a larger scale: in addition to recognizing the RNA species that target RBP binds, CLIP-seq analysis can also reveal the binding position of target RBP in each recognized RNA species.

Creative Biolabs has more than 10 years of experience in the field of RNA-protein interaction research, our existing technology provides a flexible platform for high-throughput biochemistry, which can be easily extended to any nucleic acid template to study different types of biochemical interactions. At Creative Biolabs, a wide range of biological and clinical samples are acceptable, such as cells and tissues. Based on the provided samples, we can design and complete a complete CLIP-seq analysis according to your project needs. The complete report includes raw and analytical data, charts, detailed protocols, and data analysis results. For specific analysis, please do not hesitate to contact us for more details.