Micrococcal nuclease

Dialysed virions, DBs and mock samples were treated with micrococcal nuclease (2000 Kunitz units ml⁻¹; Thermo Scientific) in the presence of 5 mM calcium chloride at 37 °C for 2 h. DNA was isolated with the QIAamp MinElute Virus Spin kit (Qiagen) without carrier RNA. RNA was isolated with the miRNeasy mini kit (Qiagen), including the DNase-I treatment or isolated with Trizol-LS reagent (Life Technologies). The DNA and RNA were also isolated from infected cells at 5 days p.i. (m.o.i. 0.1) and, uninfected cells. Next, quantified with a NanoDrop 2000, the optical density (OD) ratioat 260/280 nm was used as indicator for purity.

S. aureus cells from 30 mL culture grown in TSB medium to OD₅₅₀ = 1 were harvested by rapid centrifugation and resuspended in 390 μL ice cold 20 mM Tris lysis buffer pH 8.8, containing 10 mM MgCl₂ x 6 H₂O, 100 mM NH₄Cl, 20 mM Tris (pH 8.0), 0.4% Triton-X-100, 4 U DNase, 0.4 μL Superase-In (Ambion), 1mM chloramphenicol. Cells were disrupted by cell homogenization (FastPrep-24, MP Biomedicals) with 0.5 mL glass beads (diameter 0.1 mm) for 30 s at 6.5 m/s followed by incubation on ice for 5 min. These steps were repeated twice. To remove cell debris, cell lysates were centrifuged and subsequently stored at -80°C and 100 A₂₆₀ units of ribosome-bound mRNA fraction were subjected to nucleolytic digestion with 10 units/μl micrococcal nuclease (Thermofisher) in buffer with pH 9.2 (10 mM Tris pH 11 containing 50 mM NH₄Cl, 10 mM MgCl₂, 0.2% triton X-100, 100 μg/mL chloramphenicol and 20 mM CaCl₂). The rRNA fragments were depleted using S. aureus riboPOOL rRNA oligo set (siTOOLs, Germany) and the library preparation was performed as previously described [46 (link)].

After TOP1 inhibitor treatments, 1 × 10⁶ HCT116 cells in 35 mm dish per sample were washed with PBS and lysed with 600 μl DNAzol (Invitrogen), followed by precipitation with 300 μl 200 proof ethanol. The nucleic acids were collected, washed with 75% ethanol, resuspended in 200 μl TE buffer then heated at 65 °C for 15 min, followed by shearing with sonication (40% output for 10 s pulse and 10 s rest for 4 times). Samples were centrifuged at 20,000 × g for 5 min and the supernatants were collected and treated RNase A (100 μg/ml) for 1 h, followed by addition of 1/10 volume of 3 M sodium acetate sodium acetate and 2.5 volume of 200 proof ethanol. After 20 min full speed centrifugation, DNA pellets were retrieved and resuspended in 100 μl TE buffer for spectrophotometric measurement to quantitate DNA content. Ten μg of each sample was digested with 50 units micrococcal nuclease (Thermo Fisher Scientific, 100 units/μl) in presence of 5 mM CaCl₂, followed by SDS-PAGE electrophoresis for immunodetection of total TOP1-DPCs and ubiquitylated TOP-DPCs using specific antibodies. In addition, 2 μg of each sample was subjected to slot-blot for immunoblotting with anti-dsDNA antibody to confirm equal DNA loading.