Improved 5′ ligation reactions reduce cloning bias. (A) Schematic of the 5′ ligation splint where the DNA splint is shown in black, the 5′ RNA ligation adapter is shown in green, the randomized nucleotides are shown as 'N', and the product of the 3′ ligation (miRNA + 3′ adapter) is shown in blue. (B) Representative result of deep sequencing from an equimolar mixture of the 29 synthetic miRNAs using the original cloning method (red bars) and the thermostable RNA ligase for the 5′ ligation (black bars). Dashed line indicates expected cloning frequency. (C) Same as in (B) except the splint structure shown in (A) is used for the 5′ ligation step. (D) Scatter plot amongst replicates of miRNA-Seq done with thermo-stable RNA ligase. (E) Same as in (D) except splint ligations are being compared. (F) Scatter plot of thermo-stable RNA ligation data as a function of splinted ligation. For all plots, error bars represent standard error of the mean.