Figure 1.

Outline of high-throughput sequencing cross-linking and immunoprecipitation (HITS-CLIP), photoactivatable-ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) and cross-linking and sequencing of hybrids (CLASH) ribonomics protocols, with significant differences indicated. The steps from UV irradiation of cells through to sequencing library construction are shown. 4SU: 4-thiouridine; Ago: Argonaute. The black circles indicate mutations introduced by cross-linking damage, and the squares the nucleotide incorporated following reverse transcription. For PAR-CLIP, reverse transcription of RNA containing a crosslinked 4SU results in the misincorporation of a G in the opposite strand instead of A. The 3′ and 5′ adaptors are shown in red, and in orange following conversion to cDNA. Reproduced with permission from Sethuraman et al., Nucleic Acids Research; published by Oxford University Press, 2018, [12].