Ddpcr supermix for probe

HIV-1 RNA was quantified using the QX100™ Droplet Digital™ PCR system (Bio-Rad, Pleasanton, CA). The ddPCR mix for the usRNA assay consisted of: 10 µl 2x ddPCR™ super mix for probes (Bio-Rad); 200 nM of GAG1 and GAG2 primers; 400 nM GAG3 probe mix and 4 µl of the cDNA into a final volume of 20 µl. The total mix was placed into the 8 channel cartridge, 50 µl of droplet generating oil was added and droplets were formed in the QX100™ droplet generator (Bio-Rad). Droplet in oil suspensions were transferred to an Eppendorf® 96 well plate (Eppendorf, Germany) and placed into the T100™ Thermal Cycler (Bio-Rad). Cycling conditions were as follows: 95°C for 5 min, followed by 40 cycles of 95°C for 15 sec and 58°C for 60 sec. The ddPCR mix for the msRNA assay consisted of 10 µl 2x ddPCR™ super mix for probes (Bio-Rad); 250 nM of ks1 and mf83 primers, 500 nM of ks2-tq, and 4 µl of the cDNA into a final volume of 20 µl. PCR cycling conditions were the same as for the usRNA assay, except the annealing temperature which was 60°C. The droplets were subsequently read automatically by the QX100™ droplet reader (Bio-Rad) and the data was analyzed with the QuantaSoft™ analysis software 1.3.2.0 (Bio-Rad).

HIV-1 and CD4 RNA was quantified using the QX100 Droplet Digital PCR system (Bio-Rad, Pleasanton, CA). The HIV-1 ddPCR mix consisted of: 10 μl 2x ddPCR super mix for probes (Bio-Rad); 500 nM of Forward (5’-CATGTTTTCAGCATTATCAGAAGGA-3’) and Reverse (5’-TGCTTGATGTCCCCCCACT-3’) primers; 250 nM probe mix (5’-FAM-CCACCCCACAAGATTTAAACACCATGCTAA-TAMRA-3’) and 3 μl of the cDNA into a final volume of 20 μl. The CD4 ddPCR mix consisted of: 10 μl 2x ddPCR super mix for probes (Bio-Rad); 500 nM of Forward (5’-AGTCCTCACACAGATACGCC-3’) and Reverse (5’-ACTCACATCCGAACACTAGCAA-3’) primers; 250 nM probe mix (5’-FAM-TGAAGTGGAGGACCAGAAGGAGGA-TAMRA-3’) and 3 μl of the cDNA into a final volume of 20 μl. The total mix was placed into the 8-channel cartridge, 70 μl of droplet generating oil was added and droplets were formed in the QX100 droplet generator (Bio-Rad). Droplet in oil suspensions were transferred to a 96 well plate and placed into the T100 Thermal Cycler (Bio-Rad). Cycling conditions were as follows: 95°C for 10 min, followed by 45 cycles of 94°C for 30 seconds and 60°C for 60 seconds. Subsequently, the droplets were automatically read by the QX100 droplet reader (Bio-Rad) and the data was analyzed with the QuantaSoft analysis software 1.3.2.0 (Bio-Rad).

The expressions of selected miRNAs from microarray were measured using ddPCR. Briefly, commercial Taqman probes of selected miRNAs were produced by ThermoFisher Scientific (Rodano MI, Italy). Specific reverse transcription of miRNA was performed using TaqMan™ MicroRNA Reverse Transcription Kit (ThermoFisher Scientific, Rodano MI, Italy). The preparation of ddPCR samples was performed according to the manufacturer’s protocol for ddPCR supermix for probes (Bio-Rad Laboratories, Inc., CA, USA). After the 96-well plate was loaded on and read by a QX200 Droplet Reader and the data were collected using QuantaSoftTM (Bio-Rad, Hercules, CA). U6 small nuclear RNA (snRNA) was selected as an internal normalizer RNA.

Primers and TaqMan probes are described in Supplementary Table 4. ddPCR samples for Uc.63+ were prepared with a 20-μL reaction mixture containing 10 μL ddPCR Supermix for Probes (no dUTP, Bio-Rad), 500 nM of each forward primer and reverse primer, and 250 nM probe (FAM), and synthesized cDNA droplets were generated with 70 μL of oil using a QX100 droplet generator (Bio-Rad). Amplification was performed at 95°C for 10 min, followed by 40 cycles at 94°C for 30 s and 40 cycles at 57°C for 1 min using a C1000 Touch thermocycler (Bio-Rad). After amplification, the droplets were read on a QX100 droplet reader (Bio-Rad) and analyzed with QuantaSoft software V1.7.4 (Bio-Rad). The QuantaSoft software measured the number of positive versus negative droplets. Their ratio was then fitted to a Poisson distribution to determine the copy number of the target molecule as copies/μL.

Digital droplet PCR was performed as previously described (Henrich et al., 2012 (link)). Genomic DNA was extracted from primary non-activated CD4+ T cells using the Gentra Puregene kit (Gentra) following the manufacturer’s instructions. For each PCR reaction, 5 units of restriction enzyme BsaJI (NEB) was directly mixed with 300ng of DNA, ddPCR Supermix for Probes (Bio-Rad), and final concentrations of 900nM primers and 250nM probe. Primers/Probes were: RPP30 – forward primer GATTTGGACCTGCGAGCG, reverse primer GCGGCTGTCTCCACAAGT, probe VIC-CTGAACTGAAGGCTCT-MGBNFQ; HIV-gag – forward primer TACTGACGCTCTCGCACC, reverse primer TCTCGACGCAGGACTCG, probe FAM-CTCTCTCCTTCTAGCCTC-MGBNFQ. Droplets were prepared using the QX100 Droplet Generator (Bio-Rad) following the manufacturer’s instructions. Sealed plates were cycled using the following program: 95°C for 10 min; 40 cycles of 94°C for 30 s, 60°C for 1 min; and 98°C for 10 min, with 2°C/sec ramping speed to ensure even droplet heating. Reactions were analyzed using the QX100 Droplet Reader and number of template molecule per μl of starting material was estimated using the Quantalife ddPCR software. We aimed to run 8 replicates of ddPCR per sample – depending on the amount of DNA availability. We consistently applied a pre-determined exclusion criterion to outliers that deviated from mean values by > 2x the standard deviation.