Fig. 1.

a Overview of CLASH and b modified iPAR-CLIP methods for the formation of miRNA-target chimeras. CLASH begins with trimming of unprotected RNAs in UV crosslinked lysates with RNase and denaturation of the AGO-miRNA-target RNA tertiary complex. In modified iPAR-CLIP, the sample (C. elegans worms, for example) must be incubated with 4-thiouridine (4sU) for RNA incorporation to enhance UV crosslinking. Both CLASH and modified iPAR-CLIP protocols phosphorylate the 5′ end of the target RNA, which is then ligated to the miRNA using an exogenous RNA ligase. Subsequent 3′ end phosphatase treatment prepares the RNA for linker ligation. In CLASH, the 3′ linker is added during the “on-bead” biochemical steps, whereas in modified iPAR-CLIP, the 3′ linker is added after RNA isolation. The majority of the reads generated from CLASH and modified iPAR-CLIP are not chimeric