TILDA

Either the whole cell or RNA input was distributed in a PCR plate along with one-step RT-PCR master mix (Taqman Fast Virus 1-Step Master Mix, ThermoFisher), and the previously reported TILDA primers. The sequences used were identical to those of the original TILDA assay, which were adapted from Pasternak et al. (2008) (tat1.4: 5’-TGG CAG GAA GAA GCG GAG A-3’; rev: 5’-GGA TCT GTC TCT GTC TCT CTC TCC ACC-3’). Based on the input, pre-amplification included: reverse transcription (50 °C for 15 min), denaturation (95 °C for 2 min), and 24 cycles of amplification. Unless otherwise noted in initial characterization experiments, when using whole cells, amplification was performed using 95 °C for 15 sec and 60 °C for 4 minutes; when using a RNA input, amplification was reduced to 95 °C for 15 sec and 60 °C for 30 sec. This was done in a real time Light Cycler 480 II (Roche). Following pre-amplification, 40uL of TE buffer was added to each well and 1 µL was transferred into the next tat/rev PCR reaction. For all whole cell versus ESP RNA experiments, the first reaction was performed on separate plates due to differing thermocycling conditions. However, for the detection plate, both sample sets were run on the same plate to better allow for comparisons. For the detection plate, identical to the published TILDA, 5 µL of the Lightcycler Probe Master Mix (Roche), 0.2 µL of each 20 µM primer (tat2 and rev), 0.2 µL of the probe HIV FamZen at 5 µM and 3.4 µL of nuclease-free water for a 10 µL final reaction volume. Primer sequences and probes were synthesized by IDT and taken from the original TILDA publication which were adapted from Pasternak et al., 2008 (tat2: 5’ ACA GTC AGA CTC ATC AAG TTT CTC TAT CAA AGC A -3’; probe: 5’-/56-FAM/TTC CTT CGG /ZEN/GCC TGT CGG GTC CC/3IABkFQ/-3’). Amplification and detection was performed in a real time Light Cycler 480 II (Roche) (Preincubation 95 °C for 10 min, 45 cycles of 95 °C for 10 sec, 60 °C 30 sec, 72 °C for 1 sec followed by cooling step of 40 °C for 30 sec). Fluorescent signal was measured every cycle. Positive events were identified based on the second derivative maximum function using the Lightcycler 480 II. The cycle threshold for each positive well was then used for comparison (cycle threshold called using the LightlyCycler 480 Software, Second Derivative Maximum Function).