Sepmate 15

At the defined time points, retro-orbital blood was collected from mice. The isolation of PBMCs and plasma was achieved via centrifugation using SepMate-15 and Lymphoprep gradient medium (StemCell Technologies). 200 µl blood was immediately diluted with 800 µl PBS with 2% FBS. The blood diluent was then added to SepMate-15 tubes with 6 ml Lymphoprep (StemCell Technologies). Centrifugation at 1200 × g for 20 min was used to isolate RBCs, PBMCs and plasma. 250 µl diluted plasma was collected from the surface layer. The remaining solution at the top layer was poured to a new tube to isolate PBMCs, which were washed once with PBS + 2% FBS. The separated plasma was used in ELISA and neutralization assay.

Blood was collected in EDTA-K2 tubes vacutainers (BD and Company, Cat. No. 367863). PBMC isolation was performed at room temperature until red blood cell (RBC) lysis. Equal volumes of DPBS without Ca2⁺ or Mg2⁺ (Gibco ThermoFisher Scientific, Cat. No. 14190094) and 2% FBS (Gemini Bio, Cat. No. 100-106) was added to the whole blood and mixed (i.e. 5 mL of whole blood was diluted with 5 mL of buffer). The manufacturer’s instructions were followed in order to isolate PBMCs using Lymphoprep (StemCell Technologies, Cat. No. 07851) and either a Sepmate-15 (StemCell Technologies, Cat. No, 85415) or a Sepmate-50 (StemCell Technologies, Cat. No, 85450) depending on the blood volume. Once the PBMCs were isolated, the cells were washed with buffer two times, once at 300 × g for 8 min and once at 120 × g for 10 min with no brake. After removing the wash buffer, RBCs were lysed using 4 mL of room temperature ACK lysis buffer (Quality Biological; Cat. No. 118-156-101) for 2 minutes on ice and then the ice cold DPBS was added to the 14 mL mark and mixed. Cells were pelleted by centrifugation, 400 × g for 5 min at 4 °C. The buffer was removed and 0.5–1 mL fresh, ice cold DPBS without Ca2+ and Mg2+ was added and the cells were suspended. Cells were counted and delivered to the UAB Single Cell core, and scRNA-seq was performed.

Blood was collected in EDTA-K2 tubes vacutainers (BD and Company; Franklin, NJ). PBMC isolation was performed at room temperature until red blood cell (RBC) lysis. Equal volumes of DPBS without Ca2+ or Mg2+ (Gibco^™ ThermoFisher Scientific; Grand Island, NY) and 2% FBS (Gemini Bio; West Sacramento, CA) was added to the whole blood and mixed (i.e. 5 mL of whole blood was diluted with 5 mL of buffer). The manufacturer’s instructions were followed in order to isolate PBMCs using LymphoprepTM (STEMCELL Technologies; Vancouver, BC) and either a Sepmate^™-15 (StemCell Technologies; Vancouver, BC) or a Sepmate^™-50 (StemCell Technologies; Vancouver, BC), depending on the blood volume. Once the PBMCs were isolated, the cells were washed with buffer two times, once at 300xrcf for 8 minutes and once at 120xrcf for 10 minutes with no brake. After removing the wash buffer, RBCs were lysed using 4 mL of room temperature ACK lysis buffer (Quality Biological; Gaithersburg, MD) for 2 minutes on ice and then the ice cold DPBS was added to the 14 mL mark and mixed. Cells were pelleted by centrifugation, 400xrcf for 5 minutes at 4°C. The buffer was removed and 0.5–1 mL fresh, ice cold DPBS without Ca2+ and Mg2+ was added and the cells were suspended. Cells were counted and delivered to the UAB Single Cell core, and scRNA-seq was performed.

SW13, HEK293T, Vero E6 (all from ATCC), Huh7, HUVEC (provided by National Virus Resource Center, Wuhan, China) and DLD1 (provided by Dr. Youjun Li, Wuhan University) cells were cultured at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin (Hyclone). The primary mouse hepatocytes and MLF cells were isolated and cultured as previously described.^{55 (link),56 (link)} Primary human PBMCs were isolated from whole blood of healthy donors with SepMate™-15 (STEMCELL Technologies) according to the manufacturer’s instructions and cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin-streptomycin. The isolation of human PBMCs and its application for virus infection were approved by Institutional Review Board, Wuhan Institute of Virology, Chinese Academy of Sciences (Approval Number: WIVH31202301). FreeStyle 293F cells (provided by Dr. Bing Yan, Wuhan Institute of Virology, Chinese Academy of Sciences) were cultured at 37 °C in SMM 293-TII Expression Medium (SinoBiological) on an orbital shaker platform rotating at 120 rpm. All cell lines were tested and found to be free of mycoplasma contamination using the MycoBlue Mycoplasma Detector kit (#D101, Vazyme).

Using a needle and syringe, blood (5 mL) was collected from a peripheral vein by routine venipuncture and immediately transferred to K₂EDTA vacutainers. Blood was mixed with an equal volume of 2% fetal bovine serum (HyClone) in phosphate buffered saline (HyClone), and transferred to a barrier tube (SepMate‐15, StemCell technologies) prefilled with 4.5 mL of density gradient medium (Lymphoprep 1.077, StemCell technologies). After centrifugation at 1200 g for 15 min at room temperature, the supernatant was collected and washed three times with 10 mL of 2% fetal bovine serum in phosphate buffered saline by centrifugation at 300 g for 10 min at room temperature. Based on a hemocytometer count, cells were resuspended at a concentration of 1 × 10⁶ per mL in fetal bovine serum with 10% DMSO. After 25 min incubation at room temperature, the cells were transferred to a −80°C freezer within a Styrofoam container. Samples were held at −80°C for a maximum of 4 days before shipping on dry ice. Once arriving in Seattle, samples were rapidly thawed at 37°C for 60 s, a small volume was stained with Trypan Blue, and then counted using a hemocytometer to obtain cell concentration and viability estimates. Samples were then immediately distributed into aliquots for downstream analyses, including ATAC‐seq, RRBS‐seq, and flow cytometry analysis.

A fresh blood sample from a UC patient were obtained through UCLA Pathology and isolated PBMCs with SepMate-15 (#85415, Stemcell Technologies). A vial of UC-PBMCs (#70051) was purchased from Stemcell Technologies. The baseline characteristics of UC-PBMC are shown in Table 1. The UC-PBMC was cultured in RPMI1640 medium with 10% fetal bovine serum and 1% penicillin–streptomycin. Secreted human interleukin 8 (IL-8) was detected by DuoSet ELISA kit (DY208) from R&D Systems.