Ultraviolet (UV) Cross-Linking of Live Cells, Lysate Preparation, and RNase Titration for Cross-Linking Immunoprecipitation (CLIP)

Abstract

One of the great advantages of RNA CLIP (cross-linking immunoprecipitation) is that RNA–protein complexes can be “frozen” in situ in live cells by ultraviolet (UV) irradiation. This protocol describes UV cross-linking of mammalian tissue culture cells or whole tissues. For the latter, the tissue is typically triturated to allow UV penetration. However, depending on the thickness of the chosen tissue, this may not be necessary. It is preferable to handle the tissue as little as possible, to keep it in ice-cold buffers, and to cross-link as soon after the time of collection as is feasible to preserve native interactions at the time of cross-linking. This protocol also describes cell lysis following cross-linking, as well as treatment with RNase to partially hydrolyze the bound RNA. The first time this protocol is performed, a pilot experiment should be performed to determine the optimal RNase concentration for the particular sample. Once the RNase conditions are optimized this section of CLIP protocol can be repeated on experimental samples before proceeding through the rest of the protocol.