Fig. 3

Removal of reads derived from mispriming events eliminates artifactual peaks. Occurrences of the first six bases of the 3′ adaptor (allowing for one mismatch) (a) or bases 4–9 of the 3′ adaptor (b) in 200 bp windows around peak centers plotted using 20 bp sliding windows (with a 6 bp shift between each window) relative to the expected frequency of each adaptor-complement (calculated using 1 x 10⁶ randomly sampled exonic sequences of 200 bp). When using the original HITS-CLIP protocol (emerald), significant mispriming is observed. The use of a nested RT primer reduces the overrepresentation of the adaptor sequence (vermilion, a), but results in overrepresentation of the sequence complementary to the 3′ end of the RT primer (vermilion, b). Finally, using a nested RT primer and protected PCR primer results in much more even representation of both the adaptor sequence (blue, a) and RT primer complement (blue, b), with only modest overrepresentation of each sequence around the center of the peak