Validation of labeling efficiency. (*A*) Effect of DMSO. Five synthetic miRNAs containing different sequences were either directly labeled (blue) at 0%, 15%, or 25% DMSO or heated prior to labeling (red) at 0%, 15%, 25%, or 30% DMSO. At 25% DMSO, high labeling yields were observed independent of heat denaturation. Fifty-five separate reactions are shown. (*B*) Evaluation of dye bias. Box plots of labeling efficiencies of 53 different synthetic miRNAs labeled individually with Cy5-pCp and Cy3-pCp are shown. Results from 220 miRNA ligations are shown (see Supplement 1 for all detailed ligation data). The mean yield and standard error was 80±2 for Cy3 and 81±1 for Cy5. ANOVA *p* value of the Cy3 and Cy5 labeling efficiency distributions was 0.70, showing no difference between the labeling efficiencies of the two dyes. (*C*) Evaluation of sequence bias. Box plots of ligation efficiencies of the 220 reactions from *B* categorized according to 3′ nucleotide are shown. The mean percent yields of the labeled product and the standard errors were 82 ± 3 for A, 86 ± 2 for C, 78 ± 2 for G, and 78 ± 2 for U. ANOVA *p* value distinguishing the four distributions was 0.015, indicating that the labeling bias, while slight, is statistically significant. Ligations in *A* and *B* with low yields (<60%) were not reproducible, as subsequent ligations generally resulted in significantly higher yields (>80%). The reasons for these occasional instances of low yields are unclear. Infrequency and lack of reproducibility suggest such low yields are atypical experimental variations. High yields (>90%) were very reproducible.

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