Figure 2. Good Practices for CLIP and Related Methods. A. Testing a range of UV irradiations is recommended to find the minimal dose at which enough molecules of the RBP of interest are cross-linked to their RNA targets. No visible signal should be observed when the T4 PNK treatment is omitted as radiolabelled phosphate groups would not be added to the transcripts in the RNPs. B. The migration pattern of an RNP (e.g., PNPase) should display a smear reflecting the different RNAs to which the RBP is bound. Accordingly, the signal should be unaffected by DNase treatment. However, digestion of cross-linked RNPs with RNases should trim bound transcripts to a length corresponding to the part of the sequence that is shielded by the RBP during digestion. Consequently, the signal of a professional RBP resolves upon RNase treatment. Still, it is possible that some unconventional RBPs (e.g., SodM) could be bound to small transcripts and, thus, remain unaffected by treatments with nucleases. C. Sequencing cDNA libraries derived from control samples allows quantification of background signal and the subsequent identification of real RNA binding sites. Control samples could be a CLIP experiment on a strain in which the gene of interest was knocked-out, an untreated sample, a CLIP experiment without antibodies or unrelated antibodies or (in the case of CRAC) an untagged strain.