Post-transcriptional regulation contributes to the ability of cells to modify gene expression when facing changing external or internal environment. RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression.
CLIP (cross-linking immunoprecipitation) is a method used to analyze RNA-protein interactions or locate precise RNA modifications. This technique is performed by immunoprecipitation with an antibody against the protein-of-interest after UV cross-linking of the RNA-protein complex. CLIP-Seq, which is also known as HITS-CLIP, combines CLIP with high-throughput sequencing for identification of binding sites on RBPs. CLIP-Seq can be used to map RNA-binding landscape of interacting proteins or RNA modification sites on a genome-wide scale, which helps facilitate the understanding towards post-transcriptional regulatory networks and mechanisms.
Basic Principle of CLIP
The covalent bonds formed between proximal proteins and RNA upon exposure to ultraviolet light only occur at the sites of direct contact and preserve RNA-protein interactions.
Profacgen is capable of providing CLIP-Seq service for our customers to facilitate their research in the post-transcriptional regulatory networks. Our newest irCLIP-Seq technology utilizes infrared fluorescent dye, which took the place of traditional radioactive materials in the labeling step, enabling a broader application area. Besides, we introduced thermostable group II intro reverse transcriptase for cDNA synthesis, which improves the experimental efficiency and leads to a more simplified technical procedure.
Workflow of CLIP-Seq at Profacgen
Our extensive experience and capability can help design, develop, optimize and troubleshoot for all stages regarding your CLIP project with highest flexibility and efficiency.
Application of CLIP-Seq
|Analyze RNA-protein interaction mechanism|
|Analyze IncRNA/circRNA functional mechanism|
|Identify potential miRNA target(s)|
Sample Submission and Data Readout
|UV cross-linked cell sample is recommended for its stability (10^7 cells is preferred). Live cells in culture medium is also acceptable.|
|Sequencing analysis will be Single End 50 cycle, 20-40M.|
|A comprehensive set of data analysis includes quality control of reads, sequence alignment, DNA element analysis, Reads allocation in transcriptome, Reads allocation around TSS/TTS/Start Codon/Stop Codon, PEAK annotation, GO (gene ontology) annotation and classification, KEGG pathway analysis, etc.|
Please contact us for more details of our CLIP-Seq services. Our experts will provide and help design an optimal solution for your project and trouble-shoot for you throughout the whole process.
Huppertz I, Attig J, D’Ambrogio A, et al. iCLIP: Protein–RNA interactions at nucleotide resolution. Methods (San Diego, Calif). 2014;65(3):274-287. doi:10.1016/j.ymeth.2013.10.011.