Rnasin

To prepare the homogenization buffer, 2.5 mL of 1 M Tris (pH 7.4; ThermoFisher Scientific, #17926), 2.5 mL of 2 M KCl (RNase free; Fisher Scientific, #AM9640G), 600 μL of 1 M MgCl2 (RNase free; Fisher Scientific, #AM9530G) and 500 μL of NP-40 (ThermoFisher Scientific, #28324) were added to a 50 mL Falcon tube. The volume was made up to 50 mL with DNAse/RNAse free water and vortexed until all components were dissolved. On the day of the experiment, homogenization buffer supplemented (HB-S) was prepared by adding 5 μL of 1 M DTT (VWR, #97061-340), 50 μL of protease inhibitor (VWR, #97063-970), 25 μL of RNasin (VWR, #PAN2615), 100 μL of Cycloheximide (5 mg/mL; Fisher Scientific, #AC357420050), and 50 μL of Heparin (100 mg/mL; Fisher Scientific, #BP252420) in a 15 mL Falcon tube. The volume was made up to 5 mL with HB buffer. The HB-S solution was prepared fresh just prior to homogenization.

High salt buffer was prepared by adding 2.5 mL of 1 M Tris (pH 7.4; ThermoFisher Scientific, #17926), 7.5 mL of 2 M KCl (RNase free; Fisher Scientific, #AM9640G), 600 μL of 1 M MgCl2 (RNase free; Fisher Scientific, #AM9530G), and 500 μL of NP-40 (ThermoFisher Scientific, #28324) in a 50 mL Falcon tube. The final volume was brought to 50 mL with DNase/RNase free water and vortexed. The high salt buffer supplemented (HSB-S) was prepared before the washing step by adding 40 μL of cycloheximide (5 mg/mL; Fisher Scientific, #AC357420050), 10 μL of protease inhibitor (VWR, #97063-970), and 5 μL of RNasin (VWR, #PAN2615), in a 2 mL Eppendorf tube and the volume made up to 2 mL with HSB. This solution was prepared fresh before the washing step.

Cells were treated with RNA Cell Lysis Buffer (Boston Bioproducts, R-108) supplemented with 1:40 dilution of RNasin (VWR, PAN2611) and incubated for 5 minutes at room temperature. qRT-PCR reaction was prepared using TaqMan Fast Virus 1-Step Master Mix (Life Technologies, 4444436) according to manufacturer's protocol. For a final volume of 10 μL, 1 μL of template was added to the 384-well plate (Applied Biosystems, 4309849). A PrimeTime Std qPCR Assay was used to detect PCNA with a 6-FAM/ZEN/IBFQ 5′-3′ quencher (Integrated DNA Technologies, 326107661). Human RPLPO-VIC/MGB (Life Technologies, 4326314E) was used for normalization and was plexed with PCNA probe for all samples. Reaction was performed using QuantStudio 6 Flex.