Rnasin

HEK 293T cells were transfected with FLAG-tagged RIG-I or DAI constructs and infected with PR8 (MOI=2) for 12 hr. Cells were disrupted in lysis buffer (50 mM HEPES, 150 mM KCl, 2 mM EDTA, 1mM NaF, 0.5% NP40, 0.5 mM DTT, protease inhibitor cocktail, 25 units RNasin), and lysates were then incubated with 30 μL/sample anti-FLAG agarose bead slurry (Clone M2, Sigma) overnight with rotation at 4°C. Beads were collected by centrifugation, washed ten times with NT2 buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM MgCl₂, 0.05% NP40), resuspended in 250 μL of DNase digestion buffer (40 mM Tris pH 8.0, 10 mM MgSO₄, 1 mM CaCl₂) and treated with 25U RNasin (Promega) and 2U DNAse I (NEB) at 37°C for 20 min. Beads were then washed and resuspended in 100 μL NT2 buffer, and treated with 4 units proteinase K at 55°C for 30 minutes. 1 mL Tri-reagent (Sigma) was added to each sample, and RNA was harvested according to the manufacturer’s instructions. RNA was eluted from beads and examined by RT-qPCR or RNA-Seq as described in extended experimental procedures.

Total RNA was isolated from GGN cultures that were lytically infected at 37°C, latently infected and maintained at 37°C, or heated to 43°C for two hours as described above. RNA was collected from cells in these wells using RNAzol RT per the manufacturer's instructions (Molecular Research Center, Cincinnati, OH U.S.A.). Samples taken from 3 wells were combined. Complementary DNA was produced from each total RNA sample, and reverse transcription polymerase chain reaction (RT-PCR) was performed using the Qiagen OneStep RT-PCR kit (Qiagen, Valencia CA U.S.A.) supplemented with RNAsin (Sigma), on an Eppendorf Mastercycler epGradientS (Eppendorf AG, Hamburg, Germany). Primers used include those identifying the LAT transcript, ICP27, and GAPDH, as described previously (25 (link)). H₂O controls were performed with each primer set to determine whether or not contamination had occurred. RT-PCR reaction products were run in separate wells on 1.25% agarose TAE-ethidium bromide gels.

Cells from tumor digested specimens were adjusted to 2×10⁶/well in 24-well plates and cultured overnight with WT1, hTERT and NY-ESO-1 PepTivator (by Miltenyi). The next day, the cells were collected and washed in wash solution prepared with 0.09% NaCl solution (Bichsel AG), with Fish Gelatin 1% (Sigma Aldrich) and RNasin 0.1% (Promega). After wash, the cells were resuspended in wash solution in the presence of human Fc Block (Miltenyi Biotech), 50nM Calcein AM (ThermoFisher) and Zombie UV Fixable Viability Kit (BioLegend). After incubation, cells were stained in wash solution in the presence of CD45 PerCP Cy5.5 (clone 2D1), CD3 BV711 (clone UCHT1), and CD8 BV650 (clone SK1) (all the Abs are from BioLegend). Cells belonging to the same tumor specimen were pooled together and sorted on a MoFlo Astrios (Beckman Coulter). Sorted cells were collected in 0.2mL PCR tubes with 10 μL PBS with 0.4% BSA (Sigma Aldrich) and RNasin 0.1%. Live cells were gated as Calcein AM positive and Zombie UV negative and further gated for CD45+CD3+CD8+ markers. Collected cells were then immediately encapsulated using 10x protocol.

L4 stage worms were washed three times with cold M9 buffer supplemented with 1 mM cycloheximide and once with lysis Buffer without RNasin and PTE/DOC. Worms were then resuspended in 450μl of cold Lysis buffer (20mM Tris pH 8.5, 140 mM KCl, 1.5 mM MgCl2, 0.5 % Nonidet P40, 2% PTE (polyoxyethylene-10-tridecylether), 1% DOC (sodiumdeoxycholate monohydrate), 1mM DTT, 1mM cycloheximide, 0.4 U/μl RNasin (Sigma)). Worms were flash frozen in liquid N2 and then crushed with mortar and pestle precooled with liquid N2 (60 strokes with each pestle). Samples were then stored on ice for 40 minutes with vortexing every 10 minutes. Samples were then spun 10 minutes at 10,000x g at 4°C. Supernatant was collected and stored at −80°C. Samples were thawed and equal OD (A254) was brought to equal volume with lysis buffer and then layered on 10% to 50% (w/v) sucrose gradients. The gradients were centrifuged at 37,000 rpm for 2.5hrs at 4°C in a Beckman SW41Ti rotor, and fractionated by upward displacement through a Bio-Rad EM-1 UV monitor (Biorad) for continuous measurement of the absorbance at 254 nm using a Biocomp Gradient Station (Biocomp).