GSM7717098: Low-dose infected day 2 Hamster 2; Phodopus roborovskii; RNA-Seq
Established cell isolation protocols were modified to comply with BSL3 facility regulations. For single cell isolation, the caudal lobes of the right lung were stored in 1× PBS, 0.5% BSA containing 2 µg/ml actinomycin D. The lobes were dissociated mechanically and enzymatically. Tissue was first disrupted with forceps for 2 minutes in specific digestion medium (3.4 mg/mL collagenase Cls II (Merck), 1 mg/mL DNase I (PanReac AppliChem) in 2 mL Dispase medium per lung lobe (Corning), 50 caseinolytic units/mL) and then enzymatically digested at 37°C and 5% CO2 for 30 minutes. The cell suspension was further dissociated by pipetting, and filtered through 70 µm cell strainers. The suspensions were centrifuged at 350 g for 6 minutes at 4 °C, and the pellets were subjected to erythrocyte lysis by resuspension in appropriate buffer (BioLegend). The reaction was stopped by washing with PBS/BSA buffer and the cells were centrifuged. Cells were resuspended in low BSA buffer (1 × PBS, 0.04% BSA) and then filtered through 40 µm FloMi filters (Merck). Cell number and viability were determined microscopically using trypan blue. Sequencing libraries were generated using the 3' Chromium Next GEM Single Cell 3' Reagent kit (10x Genomics) according to the manufacturer's instructions, and sequenced on a NovaSeq 6000 device (Illumina).