Genomics v3 kit

At three days after BmNPV infection, 3 mL of hemolymph was collected from pools of twenty BmNPV-infected animals or twenty controls on ice and centrifuged at 500 g for 2 min at 4°C. The hemocyte pellet was washed twice using 1 mL of cold Grace’s insect medium (Gibco, USA) supplemented with 10% FBS (Gibco, USA), which was followed by filtering through a 40 µm cell strainer (Biosharp, China) and centrifugation at 500 g for 2 min at 4°C. The hemocyte pellet was resuspended using 200 µL PBS (calcium and magnesium-free, Gibco, USA) supplemented with 0.04% bovine serum albumin (BSA) (Solarbio, China). Cell viability and number were assessed by 0.4% trypan blue staining and cell counting using a hemocytometer. High quality hemocyte preparations were subjected to single cell encapsulation by 10X Genomics v3 kit (10x Genomics, USA).

Pooled PBMCs from blood of three ALV-J infected chickens or three control chickens at 21 DPI were, respectively, resuspended in PBS (calcium and magnesium-free; Gibco, Thermo Fisher Scientific, Waltham, MA, United States) with 0.4% bovine serum albumin (BSA; Solarbio, China), followed by passing through a 40 μm cell strainer (Biosharp, China). Cell concentration and viability were assessed using Trypan Blue and a Neubauer hemocytometer (Sigma-Aldrich, St. Louis, MO, United States). Cell viability in both samples was about 80%. Subsequently, the cell density was adjusted to 1 × 10⁶ cells/mL. High quality single cell suspensions were subjected to encapsulation using a 10x Genomics v.3 kit (10x Genomics, United States).

3×10⁷ lung cell pooled suspension was prepared from three chickens, with 1×10⁷ lung cell suspension for each treatment group. MHC Class II and CD3 positive cells were sorted out from each pulmonary cell pool after incubation with the FITC-conjugated MHC Class II and APC-conjugated CD3 antibodies (SouthernBiotech, Birmingham, AL) using Fluorescence-activated cell sorting machine (FACS Aria II, Becton Dickinson, New Jersey, USA). The gating strategy for each population is shown in S1 Fig. Equal number of MHC Class II and CD3 positive cells in each pulmonary cell pool were mixed together, followed by passing through a 40 μm cell strainer (Biosharp, China), and the cell viability was above 80%. Subsequently, the cell density was adjusted to 1 x 10⁶ cells/mL. High quality single cell suspension was subjected to encapsulation using a 10x Genomics v.3 kit (10x Genomics, USA). The library preparation and RNA-sequencing were completed by Gene Denovo (Guangzhou, China) as described previously [15 (link)]. An average of 32956 reads per cell in the H9N2 group, mean reads of 35911 per cell in the H5N1 group, and mean reads of 28419 per cell in the control group were obtained, respectively.