design: Single-cell transcriptomics of bat PBMCs with different innate immune stimulations
library_construction_protocol: 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.