Testing the usage of divalent metal cations and nucleases during NET-seq for Pol I

The goal of this study was to determine the effects of commonly used nuclease treatments and their divalent metal cation cofactors on Pol I occupancy during NET-seq. Other groups have previously used micrococcal nuclease (MNase) + CaCl2 and deoxyribonuclease I (DNase I) + MnCl2 in NET-seq protocols probing for Pol II occupancy. However, the effect of these treatments on polymerase occupancy has not been fully explored. Therefore, we tested the impact of these treatments (CaCl2, CaCl2 + MNase, MnCl2, and MnCl2 + DNase I) on Pol I occupancy in vivo. We found that the exposure of Pol I to either CaCl2 or MnCl2 caused a significant reduction in occupancy on the rDNA template. Nuclease treatment (with either MNase or DNase I) did not cause an additional significant effect on Pol I occupancy in vivo beyond that of either CaCl2 or MnCl2. Finally, we found that MnCl2 treatment caused a more significant reduction in Pol I occupancy as compared to CaCl2, suggesting that perhaps MnCl2 stimulates the intrinsic cleavage activity of Pol I more robustly vs. CaCl2 in vivo. Altogether, these findings indicate that the inclusion of these treatments (especially that of divalent metal cations) in NET-seq protocols for Pol I cause a significant change in the polymerase occupancy. Overall design: NET-seq was performed in triplicate with a WT yeast strain across five different treatment conditions: (1) Untreated Control, (2) CaCl2 Treated, (3) CaCl2 + MNase Treated, (4) MnCl2 Treated, and (5) MnCl2 + DNase I Treated.