title: GSM6098324: lung-control+; Gallus gallus; RNA-Seq
library_construction_protocol: Infected or PBS-treated chickens were sacrificed by spinal cord injury. Their lungs were subsequently perfused with PBS through the right ventricle, and were divided into two groups that were subjected to different dissociation techniques. Half of the entire pool of lungs was dissected and dissociated into single-cell suspensions using the Multi Tissue Dissociation Kit 1 (Miltenyi Biotec) in combination with a gentle magnetic activated cell sorting (MACS) dissociator (Miltenyi Biotec) and enzymatic dissociation. The other half of the lung pool was minced on ice to <1-mm cubic pieces, suspended in 5 mL of digestion buffer consisting of elastase (3 U/mL; Worthington Biochemical Corporation) and DNase I (0.33 U/mL; Sigma-Aldrich) in DMEM/F12 medium, incubated with frequent agitation at 37°C for 20 min and briefly triturated(Treutlein et al., 2014). Next, an equal volume mixture of DMEM/F12 supplemented with 10% fetal bovine serum (FBS) and penicillin–streptomycin (1 U/mL, Biological Industries) was added to the single-cell suspensions in both groups. Following enzymatic incubation, cells derived from the same lungs (after the two dissociation techniques) were passed through a 100-μm sieve and then through a 70-μm sieve, pelleted again (300 g, 5 min, 4°C), and resuspended in MACS buffer (0.5% bovine serum albumin, 2 mM EDTA in PBS). Red blood cells were removed by depletion with chicken red blood antibody (Fitzgerald Industries International, Inc.) against fluorescein isothiocyanate conjugated to magnetic beads (Miltenyi Biotec). Cell populations were sorted using chicken CD45 antibody (SouthernBiotech) against biotin conjugated to magnetic beads (Miltenyi Biotec).Chicken embryo fibroblast cell lines DF-1 (ATCC, CRL-12203) were seeded in a 15-cm plate and were cultured overnight. When the cell density reached approximately 80% confluence, cells were infected with the highly virulent Herts/33 strain at a multiplicity of infection (MOI) of 1 and were incubated at 37°C with 5% CO2 for 1 h. The growth medium was replaced with DMEM supplemented with 2% FBS and was incubated for 12 h before harvesting. There was an obvious cytopathic effect on DF-1, which confirmed NDV infection. Uninfected cells were considered negative controls. Single-cell suspensions were prepared based on the 10x Genomics protocols. Cells isolated from each group were captured in droplet emulsions using the 10x Genomics chromium single-cell instrument, and libraries were prepared using the 10x Genomics 3′ Single Cell v2 protocol for the DF-1 cell line group and the 10x Genomics 3′ Single Cell v3 protocol for the chicken group as previously described. All 10x libraries were pooled and sequenced on the NovaSeq 6000 platform (Illumina).