3′-Linker Ligation and Size Selection by SDS-PAGE for Cross-Linking Immunoprecipitation (CLIP)

Abstract

This protocol describes the purification by denaturing polyacrylamide gel electrophoresis of RNA linkers for cross-linking immunoprecipitation (CLIP). Purification is necessary because if the 3′ linker loses the puromycin blocking group, concatemerization of the 3′ linker will occur during the 3′ linker ligation reaction. In addition, truncated linkers make bioinformatic processing of the sequencing results more difficult than it need be. Additionally, this protocol describes the treatment of coimmunoprecipitated RNA tags for CLIP with alkaline phosphatase to remove the 3′ phosphate remaining after RNase digestion. Dephosphorylation prevents intramolecular circularization of RNA during subsequent ligation to the linker. The purified RNA linker, blocked with puromycin at its 3′ end to prevent linker–linker multimerization, is then ligated to the 3′ end of the RNA tag. Removal of free linker is accomplished by performing the ligation while the RNABP:RNA complex is associated, via antibody, to protein A Dynabeads, allowing thorough washing and linker removal. Additional purification is achieved by SDS-PAGE and transfer of the size-selected RNABP:RNA complexes to nitrocellulose.