Optimization of 3′ labeling via BoMoC sequence and reaction buffers. (A) Library-wide CV and 3′ precision of miRXplore miRNA as in Figure 2B, using BoMoC WT or variant enzymes. Replicate cDNA libraries have the same color. (B) Comparison of 3′ precision under different BoMoC working-stock enzyme storage buffer conditions. Significant differences in 3′ precision (denoted by asterisks) were determined by a paired, two-sided Student's t-test for each miRNA, with P-values adjusted by Bonferroni correction. Each library was benchmarked against a library prepared with enzyme diluted in 15 mM DTT in pH 6.0 diluent buffer. (C) Comparison of 3′ precision using different BoMoC proteins, incubation times, and ddNTP(s) for 3′ labeling. Significant differences were measured as described in (B).
