Pikoreal 96 machine

Viral genome copy number was quantified on a PikoReal 96 machine (Thermo Scientific) using a standard protocol (95 °C 2 min; 40 cycles of 95 °C 10 s, 60 °C 20 s, 72 °C 20 s). Each quantitative PCR analysis was performed in triplicate. Nontemplate controls (water) were included in triplicates in each assay. The KAPA SYBR FAST qPCR Master Mix (2×) Universal (Kapa Bio-systems) was used, in a 10 μL final volume. For each analysis 2 μL of the diluted cDNA was used (dilution factor of 4) and specific primers VOV-1-qRT-F1 and VOV-1-qRT-R1 at a concentration of 0.25 μM each (Table S1). The specificity of the amplicons synthesized during the PCR run was ascertained by performing a dissociation curve protocol from 60 °C to 95 °C. Specific primers used for quantification are provided in Table S1 in Supplementary material.

Total RNA was isolated from MMTV breast tumor samples using RNeasy Plus Universal kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized and real time PCR was performed on a pikoreal 96 machine (Thermo Scientific) using Maxima SYBR green qPCR master mix (Thermo Scientific). Gene-specific primers were used at a final concentration of 0.5 μM and their sequences are listed in Supplementary Table S2. All qPCR assays were performed in triplicates in at least three independent experiments using 2 different samples.

Cells were harvested and resuspended in TRIzol (Thermo Fisher Scientific). RNA was extracted according to the manufacturer’s instructions, treated with TURBO DNase (Thermo Fisher Scientific) for 30 min at 37°C to remove genomic DNA, and subsequently purified. 2 µg of RNA were reverse transcribed into cDNA using SuperScript and 10 mM dNTP Mix (both Thermo Fisher Scientific) and oligodT (Sigma-Aldrich). qPCR primers were selected using Primer-BLAST (https://www.ncbi.nlm.nih.gov/tools/primer-blast/). qPCRs were performed using cDNA (1:8 dilution) and the ORA SEE qPCR Green ROX L kit (highQu) on a PikoReal 96 machine (Thermo Fisher Scientific). GraphPad Prism was used for all graphics. Primers used are listed in Table S7.