(For this optional step follow the instructions below). 4-thiouridine enhances crosslinking of some proteins. Optional 4-thiouridine pre-incubation and UV-A crosslinking could be used for certain proteins. Knockout cells or tissue, as well as non-cross-linked cells, are good negative controls, while knockdown cells are not recommended.
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One or more negative controls should be maintained throughout the complete experiment. Remove lid, place on ice and irradiate once with 150 mJ/cm 2 at 254 nm using a Stratalinker. Remove media and add ice-cold PBS to cells (eg use cells grown in a 10 cm plate for three experiments and add 6 ml PBS).ġ.2. UV cross linking of tissue culture cellsġ.1.
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Protease inhibitors (add fresh each time)ġ/500 RNase I dilutions for library preparation 1/50 RNase I dilutions to control for antibodyĤ μL 5x PNK pH 6.5 buffer Ĥ μL 4x ligation buffer Ġ.25 μL Superscript III reverse transcriptase “iCLIP: Protein–RNA interactions at nucleotide resolution.” “iCLIP -Transcriptome-wide Mapping of Protein-RNA Interactions with Individual Nucleotide Resolution.”įurther adapted from Huppertz et al. Here is a summary of the iCLIP protocol adapted from Konig et al.
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Unlike RIP, CLIP provides information about the actual protein binding site on the RNA.ĭifferent types of CLIP exist, high-throughput sequencing-CLIP (HITS-CLIP), photoactivatable-ribonucleoside enhanced CLIP (PAR-CLIP), and individual CLIP (iCLIP). This covalent bond is irreversible, allowing stringent purification conditions. Interest in RNA-protein interactions is booming as we begin to appreciate the role of RNA, not just in well-established processes such as transcription, splicing, and translation, but also in newer fields such as RNA interference and gene regulation by non-coding RNAs.ĬLIP is an antibody-based technique used to study RNA-protein interactions related to RNA immunoprecipitation (RIP) but differs from RIP in the use of UV radiation to cross-link RNA binding proteins to the RNA that they are bound to.