Fig. 3

Ago HITS-CLIP empirically identifies robust Argonaute (Ago) footprints across the neonatal (PN2) hypothalamus transcriptome. a A histogram of the mean footscore [reads per million (RPM)] of each distinct Ago footprint that aligned to RefSeq mRNAs revealed a wide range and long-tailed distribution of footscores, with a high proportion of footprints having low footscores (Q1, 24; median, 78; Q3, 233; mean, 486). Footprints with high footscores were more likely to be identified in more than one group (biological complexity [BC] ≥ 2). We focused on a subset of footprints with footscores above the 80th percentile (footscore > 621). 16,351 Ago footprints met this criterion. b A Venn diagram of the distribution of these footprints across biological groups shows that 72% were present in more than one group (BC ≥ 2). There is also a qualitative effect of estrogen on the Ago footprint population, with the M/Form group sharing more footprints with F/Veh (59% shared) and M/Veh (63%) than F/Veh and M/Veh groups share with each other (47%). Ago footprints with a mean footscore > 621 and a BC ≥ 2 were considered “robust” and used in downstream analyses (indicated by gray shading). c A histogram of the number of robust Ago footprints aligning to each of 6689 distinct mRNAs shows that 48% of mRNAs contain more than one footprint, with an average of 1.8 footprints/mRNA. Sixteen percent of targeted mRNAs contain three or more footprints