As a result, reverse transcriptase may not read through the crosslink sites, and produce cDNA ending at the crosslinked nucleotide. Meanwhile, one or more amino acids of a crosslinked RBP can remain attached to its bound RNA due to incomplete digestion of the protein. UV irradiation can result in cDNA truncations and/or mutations at the crosslink sites, which complicates the alignment of the sequencing library to the reference genome and the identification of the crosslinking sites. Reverse transcription of the RNA followed by high-throughput sequencing of the cDNA library is now often used to identify protein bound RNA on a genome-wide scale. The RBP is protease digested to allow purification of the bound RNA. After stringent washing and gel separation the RBP–RNA complex is excised. After partial RNase digestion, antibodies specific to an RNA binding protein (RBP) or a protein-epitope tag is then used to immunoprecipitate the protein–RNA complexes. Whole cells or tissues are ultraviolet irradiated to generate a covalent bond between RNA and proteins that are in close contact. UV crosslinking immunoprecipitation (CLIP) is an increasingly popular technique to study protein–RNA interactions in tissues and cells. Wang Z, Kayikci M, Briese M et al (2010) iCLIP predicts the dual splicing effects of TIA-RNA interactions.Rogelj B, Easton LE, Gireesh KB et al (2012) Widespread binding of FUS along nascent RNA regulates alternative splicing in the brain.Tollervey JR, Curk T, Rogelj B et al (2011) Characterizing the RNA targets and position-dependent splicing regulation by TDP-43.Konig J, Rot G, Curk T et al (2010) iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution.Konig J, Zarnack K, Rot G et al (2011) iCLIP-transcriptome-wide mapping of protein–RNA interactions with individual nucleotide resolution.Hafner M, Lanthaler M, Burger L et al (2010) Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP.Huppertz I, Attig J, D’Ambrogio A et al (2014) iCLIP: protein–RNA interactions at nucleotide resolution.Ascano M, Hafner M, Cekan P et al (2012) Identification of RNA–protein networks using PAR-CLIP.Milek M, Wyler E, Landthaler M (2012) Transcriptome-wide analysis of protein–RNA interactions using high-throughput sequencing.Licatalosi DD, Aldo M, Fak JJ et al (2008) HITS-CLIP yields genome-wide insights into brain alternative RNA processing.Darnell RB (2010) HITS-CLIP: panoramic views of protein–RNA regulation in living cells.Ule J, Jensen KB, Ruggiu M et al (2003) Clip identifies nova-regulated RNA networks in the brain.Keene JD, Komisarow JM, Friedersdorf MB (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs, and protein components of ribonucleoprotein complexes from cell extracts.Hellman LM, Fried MG (2007) Electrophoretic Mobility Shift Assay (EMSA) for detecting protein-nucleic acid interactions.Modic M, Ule J, Sibley CR (2013) CLIPing the brain: studies of protein–RNA interactions important for neurodegenerative disorders. ![]() Here, we describe the detailed procedure of iCLIP. This method, individual nucleotide resolution CLIP (iCLIP) circularizes cDNA to capture the truncated cDNA and also increases the efficiency of ligating sequencing adapters to the library. This is harnessed by one variant of CLIP methods to identify crosslinking sites at a nucleotide resolution.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |