Identification of candidate miRNA-titrating mRNAs in differentiating C2C12 cells. (A) Left lanes: synthetic miR-1a and miR-206 (for calibration). (M) Size marker. Right lanes: 20 µg total RNA from differentiating C2C12 cells. (B) Quantification of three biological replicates of the experiment shown in panel A for each miRNA family (mean ± standard error). (C) Experimental identification of miR-1a/miR-206 and miR-133 targets in C2C12 cells. Cells were transfected with 2′-O-Me oligonucleotides directed against miR-1a and miR-206, against miR-133, or against no murine miRNA (“anti-Ø”). mRNAs immunoprecipitated with AGO proteins were quantified by poly(A)-independent RNA-seq. (D) Identified miRNA targets for miR-1a/miR-206 (top panel) and miR-133 (bottom panel). Red: mRNAs with 3′ UTR perfect seed matches. Blue: mRNAs whose best 3′ UTR match is one of the top three enriched imperfect matches (CNATTCC, CATNCC, or CNTTCC for miR-1a/miR-206; GACCANA, GNACCAA, or GACNCAA for miR-133). (E) Free and bound miRNA concentrations were calculated from our measures, and after conceptual loss of the miRNA binding site of interest. (F) Binding sites that exert the highest titrating activity (>10% increase in free miRNA concentration if site is lost).