Dissecting placental host-pathogen interactions: Rift Valley fever virus infection in early human trophoblast stem cells
At 24 hours post-infection, single-cell preparation was performed. Briefly, cells were rinsed with PBS and treated with a dissociation buffer containing TrypLE (Invitrogen), DNase (25 µg/mL, Roche), EDTA (0.5 mM, Invitrogen), and RNase inhibitor (1 U/mL, Thermo Scientific) for 10 minutes. Dissociated cells were collected by centrifugation at 400 × g for 5 minutes at 4 °C. hTSC lines were established from isolated villous cytotrophoblasts of human single placentae. hTSC lines were maintained on human fibronectin (Sigma) pre-coated surface in advanced DMEM/F12 (Invitrogen) containing 10 mM HEPES (Gibco), 1×B-27 Supplement (Gibco), 1×Insulin-Transferrin-Selenium-Ethanolamine (ITS-X, Gibco), and 2 mM Glutamine (Gibco), 100 ng/mL recombinant human epidermal growth factor (rhEGF, R&D Systems), 3 μM CHIR99021 (Tocris), and 5 μM Rock Inhibitor (Y-27632, Santa Cruz) at 37°C with 5% CO2. hTSCs were infected with RVFV (ZH548 strain) in serum-free advanced DMEM/F12 at an MOI of 1 for 1 hour. The inoculum was then removed, and fresh hTSC culture medium was added. Part of library preparation hTSCs were fixed using the Evercode Cell Fixation Kit (PARSE Biosciences) and passed through a 30 µm filter (pluriSelect). Fixed cells were then processed, and single-cell libraries were prepared using the Evercode WT Mini v3 protocol (Parse Biosciences).