Fig. 4.

True peaks are indistinguishable from artifacts in most HITS-CLIP data. a. A CLIP peak in the Progesterone Receptor (PGR) 3′ UTR (emerald; original protocol) in MCF-7 cells is the result of mispriming on a perfect reverse-complementary match to the final 8-bases of the RT primer (highlighted in vermilion). This sequence is also complementary to miR-888, making the resulting peak appear to contain a miR-888 target. This peak disappears completely when the reads are filtered (sky blue) or a nested RT primer and protected PCR primer is used (blue; 0, 6 and 24 h after stimulation with estradiol). b. A robust CLIP peak in the c-Myc 3′ UTR is a bonafide target of miR-34b (seed complement highlighted in emerald) [32–35]. This peaks is also present when the data are filtered (sky blue) and when a nested RT primer and protected PCR primer are used (blue). Figure based on output from http://genome.ucsc.edu (hg19 assembly) [42, 43]