Turbo dnase buffer

Cell supernatants were centrifuged at 2000 × g to remove cells and debris and filtered through a 0.45-μm filter (Millipore Sigma). Filtered supernatants were treated with 100 U TURBO DNase (Thermo Fisher) per mL supernatant and supplemented to a final concentration of 1× TURBO DNase Buffer (Thermo Fisher). The DNase reaction was allowed to proceed at 37 °C for 1 h before inactivation with 10 mM EDTA (Corning) at 70 °C for 15 min. DNA was extracted using a DNEasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s protocol. Absolute quantification of genome copy number was accomplished by real-time qPCR amplification of ORF39 (KSHV) or BMRF1 (EBV) in SensiFast Lo-Rox SYBR (Bioline) with a final primer concentration of 500 nM on a QuantStudio 6 Flex Real-Time PCR System. Standard curves were created using dilutions of pCDNA4/TO-ORF39−2XStrep (a kind gift from Dr. Britt Glaunsinger) or pSG5-BMRF1 (Addgene). Primers used for qPCR are found in Supplementary Data 3. Viral titers were compared statistically using two-tailed homoscedastic Student’s T tests to obtain P values.

Phage supernatants were concentrated by centrifugation. The phage pellet was resuspended in SM buffer (100 mM NaCl, 8 mM MgSO₄·7 H₂O, 50 mM Tris-Cl (pH 7.5)) and incubated sequentially with lysozyme (50 mg mL⁻¹), TURBO DNase and TURBO DNase buffer (ThermoFisher Scientific, Waltham, MA, USA), and proteinase K (20 mg mL⁻¹). Then, 5 M NaCl and 10% CTAB/0.7 M NaCl solution were added, and samples were transferred to phase lock gel tubes (light PLG tubes, QuantaBio, Beverly, MA, USA) with an equal amount of phenol:chloroform:isoamyl alcohol (25:24:1 v/v, pH = 8.0, ThermoFisher Scientific, Waltham, MA, USA) and centrifuged. The top aqueous DNA-containing layer was left to precipitate overnight at −80 °C in 100% ice-cold ethanol and samples were then purified with the Zymo DNA Clean and Concentrator 25 kit (Zymo Research, Irvine, CA, USA). DNA concentrations were quantified with the Qubit dsDNA high-sensitivity (HS) assay kit (ThermoFisher Scientific, Waltham, MA, USA).

For each condition, total RNA was isolated from 5 × 10
⁶ cells, which were centrifuged at 300 × g for 5 min then resuspended in 2 ml TRIzol® reagent (ThermoFisher), incubated at room temperature for 10 min before adding 0.4 ml chloroform. The cell suspension was mixed thoroughly then centrifuged at 5,000 × g, 4°C for 30 min. The top aqueous layer (~800 μl) was carefully removed and replaced with an equivalent volume of isopropanol. Samples were then centrifuged at 17,000 × g, 4°C for 30 min to pellet the total RNA. Isopropanol was removed and the pellet washed with 1 ml EtOH, then centrifuged again at 17,000 × g, 4°C for 5 min. EtOH was removed and the pellet was air-dried for 15 min then resuspended in 200 μl RNase-free H
₂O. To remove contaminant DNA, 22 μl 10× TURBO DNase buffer (ThermoFisher) and 2 μl (4U) TURBO DNase (ThermoFisher) were added the RNA suspension then incubated at 37°C for 30 min. The described TRIzol®-chloroform extraction protocol was then repeated to remove the DNase, and the final RNA sample resuspended in 200 μl RNase-free H
₂O. RNA was isolated twice for each condition in two independent experiments.
ds-cDNA libraries for each sample were prepared and sequenced at the Wellcome Trust Centre for Human Genetics, Oxford, using the HiSeq
^® 4000 Sequencing System (Illumina).

Frozen tissue was ground into a fine powder using a ⅛′′ steel ball bearing with 1 min shaking at 25 Hz in a TissueLyser II (Qiagen, Hilden, Germany). Total RNA was extracted from finely ground tissue using TRIzol reagent (#T9424‐200ML; Sigma‐Aldrich, St. Louis, MO, USA) at a ratio of 1 ml solution per 100 mg ground tissue. Residual phenol was removed from the crude extract through two chloroform extractions at a ratio of 1:5 (v/v), followed by precipitation using isopropanol at 1:1 (v/v). The precipitated RNA was washed twice with 70% ethanol and resuspended in 1 mm sodium citrate buffer (pH 5.4). RNA quantification was performed through spectrophotometric analysis at 260 nm using the Nanodrop ND‐1000 Spectrophotometer, and RNA quality was assessed using 1% agarose gel electrophoresis or the LabChip GX Touch (PerkinElmer, Waltham, MA, USA). Five micrograms of purified total RNA was combined with 5 μl of TURBO DNase buffer and 1 μl TURBO DNase (Thermo Fisher Scientific, Waltham, MA, USA) in a 50 μl reaction and incubated at 37°C for 30 min. DNA nuclease‐treated RNA was then purified using 1.8× Sera‐Mag paramagnetic particles.