Qx200 autodg droplet digital pcr system

For RT-ddPCR, cDNA was synthesized from 500 ng of RNA and a 20 μL reaction mixture with the following components: 4 μL of 5× 1st strand buffer (Invitrogen, Waltham, MA, USA), 2 μL of 0.1 M DTT (Invitrogen, Waltham, MA, USA), 2 μL of random hexamers (50 μg/mL; Promega, Madison, WI, USA), 1 μL of 10 mM deoxynucleoside triphosphates (dNTPs) mix, 0.1 μL of RNaseOut (40 U/μL; Invitrogen), 0.5 μL of SuperScript II RT (200 U/μL; Invitrogen, Waltham, MA, USA) and nuclease-free water to bring the final reaction volume to 20 μL. The reverse transcription reaction was carried out at 42 °C for 50 min followed by at 70 °C for 15 min. Negative controls containing no SuperScript II RT were also included in the reaction [42 (link)]. RT-ddPCR was performed using Bio-Rad QX200 AutoDG Digital Droplet PCR system. Primer sets used for the amplification of HIV-1 mRNA are detailed in Table 4.

DdPCR was performed using the QX200™ AutoDG™ Droplet Digital™ PCR System (Bio-Rad, Hercules, CA, USA). The reaction volume of 22 µL consisted of 2× Supermix for probes (no UTP), 900 nM primers, 250 nM probes, and 9 or 10 µL of cfDNA. All reagents were purchased from Bio-Rad, and assays were either purchased from Bio-Rad or custom-designed by Applied Biosystems (Table S4). All samples were measured as triplicates as a minimum. Wet-lab validated assays were used when possible, and the remaining assays were designed by Bio-Rad or Applied Biosystems and validated in the lab. Data were analyzed using QuantaSoft v.1.7.4.0917 software (Bio-Rad). Each run contained positive and negative controls. Gene Strands (Eurofins Genomics, Ebersberg, Germany) diluted in cfDNA from anonymous blood donors were used as mutation-positive controls. The limit of detection for each assay was determined using blood samples from anonymous donors collected from the blood bank at Aarhus University Hospital, as previously described (Table S4) [39 (link),40 (link)]. All mutations chosen for ddPCR analysis were tested in a corresponding PBC sample to rule out clonal hematopoiesis. Results are depicted as both mutant allele concentration and mutant AF, as we have recently shown that the biological variation of ctDNA may affect these two metrics differently [41 (link),42 (link)].

Deletion in GRID2 was confirmed using BCM chromosomal microarray version 10.2 [12 (link)]. Droplet digital (dd) PCR for RPS6KC1 was performed using the QX200™ AutoDG™ Droplet Digital™ PCR System (Bio-Rad) following the manufacturer’s protocols and analyzed in QuantaSoft™. Detailed conditions and primers are listed in Additional file 3: Supplementary Materials and Methods.

qRT-PCR and ddPCR were performed according to the standard procedures as described previously.^{1 (link)} Sequence of the primers and probes for the SARS-Cov-2 virus was obtained from National Institute for Viral Disease Control and Prevention (http://nmdc.cn/#/nCoV). Detailed information on sequences was summarized in Supplementary Table S4. Total RNA was extracted from FFPF samples by an FFPF RNA Kit (ADx-FF04, Amoy Diagnostics Co., Ltd, China). Nucleic acid extraction of SARS-Cov-2 was used by real-time RT-PCR method with a SARS-Cov-2 Nucleic Acid Detection Kit (8.0131901X024E, Amoy Diagnostics Co., Ltd) according to the manufacturer’s instructions. Digital droplet PCR assays were performed on QX200 AutoDG Droplet Digital PCR system (Bio-Rad) with One-Step RT-ddPCR Advanced Kit (186-4021, Bio-Rad Co., Ltd).