Qx100 droplet generator

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.

Droplet digital PCR reactions consisted of 20 μL mixture per well containing 10 μL of ddPCR Probe Supermix (no deoxyuridine triphosphate, Bio-Rad, Hercules, CA, USA), 900 nM of primers, 250 nM of probe, and 5 μL of cDNA generated from either HIV-1 virion RNA or the ‘IVT’ standards. The ddPCR reactions were incorporated into droplets using the QX100 Droplet Generator (Bio-Rad). Nucleic acids were amplified with the following cycling conditions: 10 min at 95 °C, 45 cycles of 30 s at 95 °C and 59 °C for 60 s, and a final droplet cure step of 10 min at 98 °C using a Vapo.Protect Mastercycler^® (Eppendorf, Hamburg, Germany). Droplets were read and analyzed using Bio-Rad QX200 system and QuantaSoft software (version 1.7.4.0917) in ‘absolute quantification’ mode. Only wells containing ≥11,000 droplets were accepted for further analysis. No-RT controls (containing ≥10,000 cp HIV RNA) and no-template controls (NTCs) were included in every independent experiment.

Total RNA were extracted using QIAzol Lysis Reagent (Qiagen, Hilden, Germany) from TA of Col3a1^+/G182R mice and Col3a1^+/+ mice, stored at -80°C, according to the manufacturer’s instructions. Total RNA were measured (NanoDrop One, Thermo Fisher, Waltham, MA), normalized to 10ng/μL and then reverse transcribed into cDNA using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). The cDNA was used as template for the droplet digital PCR (ddPCR) analysis as follows: ddPCR reaction mixture (12.5μL ddPCR Supermix for Probes (No dUTP) (Bio-Rad, Hercules, CA), 1 μL specific primers of WT and mutated alleles (S1 Table), 20 ng cDNA, and water up to 20 μL) was used for droplet generation. PrimePCR ddPCR Expression Probe Assay: Vim, Mouse (0.5X, Bio-Rad, Hercules, CA) was used as the internal control to normalize the expression of Col3a1. After droplet generation (QX100 Droplet Generator, Bio-Rad Laboratories, Hercules, CA), 40 μL of sample was manually transferred to a 96-well plate (Eppendorf, Hamburg, Germany). Amplification was performed (40 cycles of amplification, temperature of hybridization 61°C) and fluorescent intensity was measured in a QX100 Droplet Reader (Bio-Rad Laboratories, Hercules, CA) and the signal data was analysed using QuantaSoft Analysis Pro (1.0.596, Bio-Rad Laboratories, Hercules, CA).

FRET-based RNA probe (5'-Alexa488-UCUCGGUGCGUUG-BHQ1-3'; Japan Bio Services, Saitama, Japan) in the amount of 20 μL at 200 nM concentration mixed with each bacterial dilution, 20 μL of 200 nM FRET-based RNA probe mixed with 5 ng/μL RNase A (Merck Millipore), 20 μL of 200 nM FRET-based RNA probe, or 20 μL of 200 nM FRET-based RNA probe mixed with each cell-free medium was dispensed into a sample well of a DG8 cartridge (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Then, 70 μL of 1% Pico-surf1 in Novec7500 (Dolomite, Royston, UK) was then loaded into each oil well of the DG8 cartridge, and the cartridge was covered with a DG8 gasket (Bio-Rad Laboratories, Inc.) and loaded into a QX100 droplet generator (Bio-Rad Laboratories, Inc.). Pico-surf1 dissolved in continuous oil, Novec7500, is a polyfluorinated surfactant, which stabilizes micro-droplets under a wide range of temperatures and biological conditions. The droplets in the emulsion wells of the DG8 cartridge were transferred into 1.5-mL tubes.