U1A iCLIP-seq (individual-nucleotide resolution UV crosslinking and immunoprecipitation coupled with RNA sequencing) analysis in HeLa cells

To better understand the mechanism of U1A functions in human cells, we performed U1A iCLIP-seq analysis in HeLa cells. iCLIP-seq is a previously well-established protocol that is believed to detect protein-RNA interaction at individual-nucleotide resolution. Indeed, successful completion of U1A iCLIP-seq has helped us to illuminate more detailed mechanism through which U1 snRNP prevents mRNA PCPA (premature cleavage and polyadenylation) Overall design: HeLa cells were grown in 10 cm cell dishes, protein-RNA were crosslinked by UV light exposure. U1A-RNA were immunoprecipitated by anti-U1A antibody (santa cruz : sc-101149). Immunoprecipitated RNAs were further purified and prepared for RNA-seq library construction. For detailed protocol, please check the published protocol (Methods. 2014 Feb; 65(3): 274–287.)