TABLE 1.
Impact of TCEP in enzyme storage diluent on 3′-labeling activity
| Fig. | Storage diluent | 3′-labeling BoMoC | 3′ label | Timing (m) | CV | Mean difference | CI 95% | Adjusted P-value | miRNA (n) | 3′ precision |
|---|---|---|---|---|---|---|---|---|---|---|
| 3B | pH 6.0, 0.2 mM TCEP | F753A | ddR | 90/30 | 0.87 | 1.02% | (0.84%−1.2%) | 3.98×10−24 | 902 | 0.72 |
| 3B | pH 6.0, 0.5 mM TCEP | F753A | ddR | 90/30 | 0.91 | −0.22% | (−0.42% to −0.03%) | 1.00×1000 | 899 | 0.71 |
| 3B | pH 6.0, 1.0 mM TCEP | F753A | ddR | 90/30 | 0.86 | −1.77% | (−1.97% to −1.56%) | 4.38×10−53 | 902 | 0.70 |
| 3B | pH 7.5, 0.2 mM TCEP | F753A | ddR | 90/30 | 0.94 | 1.61% | (1.4%−1.83%) | 1.26×10−41 | 900 | 0.73 |
| 3B | pH 7.5, 0.5 mM TCEP | F753A | ddR | 90/30 | 0.92 | 1.18% | (0.96%−1.39%) | 3.14×10−22 | 899 | 0.73 |
| 3B | pH 7.5, 1.0 mM TCEP | F753A | ddR | 90/30 | 1.04 | 0.65% | (0.43%−0.87%) | 5.85×10−06 | 894 | 0.72 |
| 3C | pH 7.5, 0.2 mM TCEP | F753A | ddA | 30 | 0.65 | −2.73% | (−3.73% to −1.72%) | 1.18×10−04 | 903 | 0.75 |
| 3C | pH 7.5, 0.2 mM TCEP | F753A | ddA | 60 | 0.63 | −0.51% | (−1.51%−0.49%) | 1.00×10−00 | 903 | 0.78 |
| 3C | pH 7.5, 0.2 mM TCEP | F753A | ddA | 120 | 0.57 | 1.98% | (1.01%−2.94%) | 6.08×10−2 | 903 | 0.80 |
| 3C | pH 7.5, 0.2 mM TCEP | W403AF753A | ddA | 30 | 0.64 | 0.63% | (−0.39%−1.65%) | 1.00×10−00 | 903 | 0.79 |
| 3C | pH 7.5, 0.2 mM TCEP | W403AF753A | ddA | 60 | 0.61 | 3.37% | (2.36%−4.38%) | 8.18×10−08 | 903 | 0.81 |
| 3C | pH 7.5, 0.2 mM TCEP | W403AF753A | ddA | 120 | 0.59 | 5.51% | (4.48%−6.54%) | 2.12×10−21 | 903 | 0.84 |
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For 3′-labeling time, 90/30 refers to 90 min of 3′ labeling with ddATP followed by a 30-min ddGTP-supplementation. Mean difference in 3′ precision and a 95% confidence interval were computed from a paired, two-sided Student's t-test, Bonferroni adjusted P-values. For Figure 3B, all libraries were compared to a library prepared by F753A diluted in pH 6.0 15 mM DTT diluent buffer and labeled with ddRTP for 90/30 min. For Figure 3C, all libraries were compared to a library prepared by F753A diluted in pH 7.5 0.2 mM TCEP diluent buffer and labeled with ddRTP for 90/30 min.
