RNA–Protein Crosslinking and Immunoprecipitation (CLIP) in Schizosaccharomyces pombe

The functions of RNA binding proteins can only be elucidated if their RNA targets are known. The "crosslinking and immunoprecipitation" (CLIP) of proteins and nucleic acids allows the identification of interacting RNAs in the context of an intact cell, which is important for genome-wide reconstructions of genetic circuits. In this chapter is presented a modified experimental procedure which includes in vivo crosslinking with formaldehyde, leading to the formation of a reversible bond between protein and interacting RNAs. This is followed by a partial digestion of the RNAs, stringent immunoprecipitation of the complex, and ligation of the RNA adapters. The formaldehyde crosslinks are then reversed by heat treatment and the released RNAs reverse-transcribed, amplified, and cloned into a vector for sequencing. Alternatively, the amplified cDNAs are directly sequenced, using any next-generation sequencing technology. By using a knock-out strain of Schizosaccharomyces pombe which is complemented by an HA-tagged protein of interest, the CLIP analysis for S. pombe has been optimized. If successfully established, the time requirement for this is about two weeks, followed by sequencing.