Table 1. The central features of CLIP methods from the perspective of data analysis^a.

Methods	Specificity	Resolution	Sensitivity
HTTS-CLIP, CLIP-seq, CRAC	++ to +++ Strong detergents and high-salt washes are used with further purification by SDS-PAGE and membrane transfer, which allows one to optimize RNase conditions and ensure that copurified RBPs and noncrosslinked RNAs are removed. Thus, only specific RNAs crosslinked to the immunoprecipitated RBP are normally isolated, but specificity depends on careful optimization and visualization of the purified complexes.	Oligonucleotide corresponding to the size of readthrough cDNAs	++ Limited by the loss of cDNAs truncated at crosslink sites
iCLIP, 4SU-iCLIP, FAST-iCLEP, BrdU-CLIP, irCLIP, Fr-iCLEP, sCLIP	++ to +++, as in HTTS-CLIP	Nucleotide corresponding to the start of truncated cDNAs	++ to +++ Increased due to amplification of truncated cDNAs, as well as other method-specific optimizations
eCLIP, FLASH	+ to +++ The purity of protein-RNA complexes is not validated by visualization. Blind cutting from the membrane is used in eCLEP, while SDS-PAGE separation is removed in sCLIP. Thus, copurification of nonspecific RBPs is not monitored, which could result in large variations in specificity.
iCLAP, uvCLAP	++ to +++ Expression of affinity-tagged proteins permits rigorous washing with denaturing conditions, which removes copurified RBPs. However, expression of tagged RBPs could in some cases affect RNA specificity or lead to artifacts associated with overexpression.
PAR-CLIP	++ to +++, as in HTTS-CLIP	Nucleotide corresponding to the crosslink-induced mutations	+ to +++ Limited by the loss of cDNAs truncated at crosslink sites but offset to differing degrees by increased crosslinking efficiency for some RBPs
CIMS of HITS-CLIP	++ to +++, as in HTTS-CLIP		+ Limited by the loss of cDNAs truncated at crosslink sites and the low proportion of cDNAs with deletions
RIP-seq	+ Preserves protein-protein interactions due to mild washing conditions or formaldehyde crosslinking, thus copurifying interacting RBPs	Transcript-level resolution achieved by the original version of RIP-seq, since it doesn’t fragment the bound RNAs	++ If no crosslinking is used, then transient weak interactions that take place in vivo can be lost during immunoprecipitation, and abundant RNAs tend to dominate the pulldown, leading to decreased coverage of introns or other low-abundant RNAs.
RIPiT-seq, DO-RIP-seq	++ Use of RNase reduces copurification of RBPs that bind to the same transcripts, but such RBPs can still be purified due to the preserved protein-protein interactions. Sequential IP with two separate antibodies can specify RNPs composed of multiple RBPs in RIPiT-seq.	Oligonucleotide corresponding to the size of cDNAs due to the use of RNase to fragment the bound RNAs	++ Due to saturation with reads from abundant RNAs bound by copurified RBPs, the method has limited sensitivity for RBPs that primarily bind introns and other low-abundant RNAs.

Abbreviations: +, moderate; ++, high; +++, best; 4SU, 4-thiouridine; BrdU, bromodeoxyuridine; cDNA, complementary DNA; CIMS, crosslink-induced mutation sites; CLAP, crosslinking and affinity purification; CLIP, UV crosslinking and immunoprecipitation; CRAC, crosslinking and cDNA analysis; DO, digestion-optimized; eCLIP, enhanced CLIP; FAST, fully automated and standardized; FLASH, FADD-like IL-1 (3-converting enzyme-associated huge protein; Fr, fractionation; HITS, high-throughput sequencing; iCLAP, individual-nucleotide resolution CLAP; iCLEP, individual-nucleotide resolution CLIP; IP, immunoprecipitation; irCLIP, infrared CLIP; PAR, photoactivatable ribonucleoside-enhanced; RBP, RNA-binding protein; RIP, RNA immunoprecipitation; RIPiT, RNA-protein IP in tandem; RNP, ribonucleoprotein complex; sCLIP, simphfied-platform CLIP; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; seq, sequencing; uvCLAP, ultraviolet CLAP.

^aCLIP methods are grouped according to how the reads are used to identify binding sites. Associated technical features and limitations are summarized in terms of resolution, sensitivity, and specificity. Colors represent the quality of the parameter (red is poor, orange is adequate, and green is good).