GSM5129163: UninfectedGroup_T24; Sus scrofa; RNA-Seq
Primary porcine alveolar macrophages (PAMs) were collected from 3-week-old Specific Pathogen Free (SPF) pigs, and maintained in RPMI 1640 medium (GIBCO, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, USA), 200 mg/mL streptomycin, and 200 IU/mL penicillin at 37℃. The ASFV Pig/SY18 strain was isolated and further propagated in PAMs for amplification culture. Viral titration was performed by hemadsorption (HAD) assays and expressed as HAD 50 /ml. All experiments with live virus were conducted in biosafety level 3 (BSL-3) facilities in Harbin Veterinary Research Institute (HVRI) of the Chinese Academy of Agricultural Science (CAAS). 5×105 PAM cells per well were seeded in 12-well plate and infections were carried out at a multiplicity of infection (moi) of 1. After 1 h of incubation, the viruses were removed and the cells were washed twice by PBS, then fresh medium was added. At the indicated times (0h, 2h, 6h, 10h, 15h, 24h and 36h), cells were washed gently and trypsinized. Single cell suspensions were washed with PBS + 0.04%BSA, resuspended in 200 μL PBS + 0.04%BSA, passed through a 30 μm filter, and placed on ice. Cells were counted using trypan blue exclusion on a LUNA-II™ Automated Cell Counter (Logos Biosystems). The appropriate numbers of cells to achieve a targeted cell input of 10,000 cells per condition were used to generate the GEMs (Gel Bead-In Emulsion). The Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1(10X Genomics, Pleasanton, CA, USA) was used for GEMs generation, cDNA synthesis and library preparation following the manufacturer's instruction.