title: Atherosclerosis licenses for an exceeding immune response in COVID-19 disease by interferon priming in circulating myeloid cells
design: Atherosclerosis licenses for an exceeding immune response in COVID-19 disease by interferon priming in circulating myeloid cells
library_construction_protocol: Blood was obtained from hospitalized SARS-CoV-2-infected patients with moderate COVID-19 according to the WHO Clinical Progression Scale (WHO-CPS)5 , not requiring intensive-care treatment, with (n=5) and without (n=6) preexisting atherosclerotic cardiovascular disease. ASCVD was defined as a history of myocardial infarction, severe carotid artery stenosis (> 70% narrowing) or atherosclerotic stroke, according to the European Society of Cardiology. As a comparision group patients with comparable age  and disease severity without a prior history of cardio- or cerebrovascular illness were included. Patients with end-stage renal failure, malignancies, rheumatological conditions and diseases requiring immunosuppression were not included. All patients were hospitalized for moderate COVID-19 pneumonia requiring oxygen supplementation but no invasive ventilation or intensive care unit treatment. Patients were included between July 03, 2020, and November 11, 2020.  Blood was sampled in EDTA tubes on 3 after hospitalization before vaccines against SARS-CoV-2 were available. Peripheral blood mononuclear cells (PBMCs) were isolated via density centrifugation (800g for 20 min) of PBS-diluted whole blood over Ficoll-Paque. The PBMC layer was carefully collected, washed with PBS, resuspended in 80% MetOH and frozen at -80°C. Written informed consent was obtained from all patients. The samples were acquired as part of the CAPNETZ Study, which was approved by the local ethics review board (protocol number 11/17) and complies with the Declaration of Helsinki. Cellular suspensions of thawed, MetOH-fixed PBMCs were loaded on a 10X Chromium Controller (10X Genomics) according to manufacturer's protocol based on the 10X Genomics proprietary technology. All scRNA-seq libraries were prepared using Chromium Single- Cell 3′ v3 Reagent Kit (10X Genomics) according to manufacturer's protocol. Briefly, the initial step consisted of performing an emulsion where individual cells were isolated into droplets together with gel beads coated with unique primers bearing 10X cell barcodes, unique molecular identifiers, and poly(dT) sequences. Reverse transcription reactions were engaged to generate barcoded full-length cDNA followed by the disruption of emulsions using the recovery agent and cDNA clean up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific). Bulk cDNA was amplified using a Biometra Thermocycler TProfessional Basic Gradient with 96-Well Sample Block (98 °C for 3 minutes; cycled 14×: 98°C for 15 s, 67°C for 20 s, and 72°C for 1 minute; 72°C for 1 minute; held at 4°C). Amplified cDNA product was cleaned with the SPRIselect Reagent Kit (Beckman Coulter). Indexed sequencing libraries were constructed using the reagents from the Chromium Single-Cell 3′ v3 Reagent Kit as follows: fragmentation, end repair, and A-tailing; size selection with SPRIselect; adaptor ligation; post-ligation clean up with SPRIselect; sample index polymerase chain reaction; and clean up with SPRI select beads. Library quantification and quality assessment were performed using Bioanalyzer Agilent 2100 using a High-Sensitivity DNA chip (Agilent Genomics)