Single-cell transcriptomics of bat PBMCs with different innate immune stimulations
Single-cell transcriptomics of bat PBMCs with different innate immune stimulations
Rousettous blood was collected from five individuals during transfusion and kept in heparinized tubes to prevent clot formation. peripheral blood mononuclear cells (PBMCs) were isolated using 63% percoll (Cytiva;17-0891-01) solution, according to standard density gradient centrifugation methods (30 min, 400G). Samples were then treated with RBC cell lysis (Sigma, according to the manufacturer's protocol), quenched with 10% FBS in D-PBS,and again pelleted and resuspended.  Cells were then counted and divided into 12-well plates with approximately 2x106 cells in each well. Cells were incubated overnight at 37°C in medium (IMDM (Biological Industries), supplemented with 10% of HI FBS (Rhenium, 10270106), Penicillin-Streptomycin-Amphotericin-B (Biological Industries, 1 03-033-1C)). On the following day, to profile the immediate innate immune response, cells were either stimulated with LPS 100 ng/ml (Invivogen, tlrl-3pelps), 1 μg/ml high-molecular mass poly(I:C) (Invivogen, tlrl-pic), transfected with 2 μg/ml Lipofectamine 2000 (ThermoFisher, 11668027), and either mock-transfected or or kept untreated. Following 4 hours of incubation, the stimulation was terminated by collecting the floating cells and trypsinizing the attached cells on the bottom of the well. Cells were then washed in 0.05% PBS  cells were counted and loaded on the 10X Genomics Chromium instrument for single cell sequencing, aiming to capture 10,000 cells. We used either 10x Genomics CG000390 Rev B protocol Chromium Single Cell 3′ gene expression V.3.1 (10x Genomics) for CellPlex samples, or CG000204_ChromiumNextGEMSingleCell 3' v3.1_Rev D protocol for the standard  protocol. Libraries were sequenced on an Illumina NovaSeq 6000 using the NovaSeq 6000 SP Reagent Kit v1.5 (100 cycles) to generate 84-bp paired end reads. cDNA library preparation was were carried out according to the manufacturer's protocol.