Harvest the cells by scraping with a cell lifter.Irradiate once with 150 mJ/cm 2 at 254 nm. Remove the media and add 6 ml ice-cold PBS to cells grown in a 10 cm plate (enough for three experiments).We successfully applied iCLIP to study hnRNP C particle organization on a genome-wide scale and assess its role in splicing regulation 14.ġ. Importantly, sequencing the truncated cDNAs provides insights into the position of the cross-link site at nucleotide resolution. We recently developed iCLIP (individual-nucleotide resolution CLIP), which captures the truncated cDNAs by replacing one of the inefficient intermolecular RNA ligation steps with a more efficient intramolecular cDNA circularization (Figure 1) 14. Such truncated cDNAs are lost during the standard CLIP library preparation protocol. In addition, primer extension assays indicated that many cDNAs truncate prematurely at the crosslinked nucleotide 13. This is partly due to the restricted amount of co-purified RNA and the two inefficient RNA ligation reactions required for library preparation. Recently, PAR-CLIP was introduced that uses photoreactive ribonucleoside analogs for cross-linking 11,12.ĭespite the high specificity of the obtained data, CLIP experiments often generate cDNA libraries of limited sequence complexity. In combination with high-throughput sequencing technologies, CLIP has proven as a powerful tool to study protein-RNA interactions on a genome-wide scale (referred to as HITS-CLIP or CLIP-seq) 9,10. CLIP combines UV cross-linking of proteins and RNA molecules with rigorous purification schemes including denaturing polyacrylamide gel electrophoresis. In order to increase the specificity and positional resolution, a strategy referred to as CLIP (UV cross-linking and immunoprecipitation) was introduced 7,8. These approaches were prone to identifying indirect or non-physiological interactions 6. Initial attempts to study protein-RNA complexes in their cellular environment employed affinity purification or immunoprecipitation combined with differential display or microarray analysis (RIP-CHIP) 3-5. Protein-RNA interactions can be studied using biochemical methods, but these approaches do not address RNA binding in its native cellular context. Therefore, an essential step towards understanding transcript regulation at the molecular level is to gain positional information on the binding sites of RBPs 2. The unique composition and spatial arrangement of RNA-binding proteins (RBPs) on a transcript guide the diverse aspects of post-transcriptional regulation 1.
0 Comments
Leave a Reply. |
Details
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |