In eukaryotes, RNA Binding Protein (RBP) exists in a wide variety of categories, and plays an important role in multiple post-transcriptional regulation processes such as RNA cleavage, transport, sequence editing, intracellular localization and translational control, hence the function of RBP is of great significance in addressing human diseases. RNA Immunoprecipitation (RIP), a technique for studying the binding of RNA and proteins in cells, is a powerful tool for understanding the dynamic processes of post-transcriptional regulation networks. The corresponding RNA-protein complex is precipitated by using an antibody against the target protein, and the RNA bound to the complex is available for sequencing analysis after separation and purification. The application of RIP is expected to discover a variety of RNA regulatory targets.
● Whole transcriptome coverage: screening and identification of protein binding sites in the whole transcriptome range
● High sensitivity: millions of sequence tags are available for each sample to detect more protein binding sites on the transcript
● High accuracy: high level of signal-to-noise ratio can be obtained to accurately distinguish between real signals and noise
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1. What is the difference between CLIP-Seq and RIP-Seq?
Both are techniques for detecting the interaction of RNA with RNA binding proteins, and their major difference lies in the gel purification process after UV crosslinking and precipitation of complex. CLIP-Seq can mark radioactive or non-radioactive labels on the RNA ends of RNA-protein complexes and separate these complexes in SDS-PAGE, thus increasing the specificity and improving the quality of cDNA libraries.
2. What sequencing methods are recommended for CLIP-Seq and RIP-Seq?
CLIP-Seq generally uses the SE50 sequencing mode, because the RNA fragments obtained by CLIP are generally small in length. The RNA fragment obtained by RIP is relatively longer, so the SE50 or PE150 sequencing modes are both optional according to the actual quality inspection requirement and research purpose. In short, the sequencing strategy depends mainly on the length of RNA after IP.