Table 2 |.
Features of selected CLIP methods
| Method | Advantages | Disadvantages | References |
|---|---|---|---|
| HITS-CLIP/PAR-CLIP | Well-established method; can identify interactions at the 3′ end of RNA | Long protocol; UV cross-linking may be poor; reverse transcriptase must bypass the cross-linked nucleotide | 62,65 |
| iCLIP | Well-established method; reverse transcriptase does not need to bypass the cross-linked nucleotide | Long protocol; UV cross-linking may be poor; interactions near the 3′ end of an RNA may be unidentifiable because reverse transcriptase stops at the cross-linked nucleotide | 77 |
| eCLIP | Avoids the circularization step of iCLIP, which can be an unreliable reaction | Long protocol; UV cross-linking may be poor; must ligate a single-stranded DNA adaptor to single-stranded cDNA | 78 |
| irCLIP | Uses a fluorescent adaptor to visualize cross-linked RNA at each step; efficient | Long protocol; UV cross-linking may be poor; interactions near the 3′ end of an RNA may be unidentifiable because reverse transcriptase stops at the cross-linked nucleotide | 79 |
| HITS-CLIP variants 1 and 2 | Short protocol; increased reliability because both ligations are carried out on-bead | UV cross-linking may be poor; reverse transcriptase must bypass the cross-linked nucleotide | 67,74–76 |
| GoldCLIP | No gel purification step; short protocol | UV cross-linking may be poor; must express a fusion protein; interactions near the 3′ end of an RNA may be unidentifiable because reverse transcriptase stops at the cross-linked nucleotide | 81 |
| fCLIP | Replaces UV cross-linking with formaldehyde, which may have a higher efficiency for proteins binding double-stranded RNA | Long protocol; formaldehyde cross-linking for CLIP is not as well understood as standard UV cross-linking | 27 |
| BrdU-CLIP | BrdU in reverse transcription removes ‘empty adaptor’ reads that can clutter HITS data | Long protocol; UV cross-linking may be poor; interactions near the 3′ end of an RNA may be unidentifiable because reverse transcriptase stops at the cross-linked nucleotide | 80 |
| TRIBE | No need to purify the protein of interest; no dependence on UV cross-linking; RBP interaction may occur anywhere in RNA | Less extensive examples of effective use of the method | 90 |
| RNA tagging | No need to purify the protein of interest; no dependence on UV cross-linking; straightforward protocol | Has not been demonstrated outside Saccharomyces cerevisiae; might not work on RBPs distant from the 3′ end of RNA | 91 |
Advantages and disadvantages of different methods of identifying RNAs bound to a protein of interest. Only a selected subset of CLIP methods is included, and many other excellent protocols exist.