Single-cell transcriptomics of mouse PBMCs with diffrent innate immune stimulations
Single-cell transcriptomics of mouse PBMCs with diffrent innate immune stimulations
Since the amount of blood from each individual is limited, we combined blood from several individuals (6 and 9 in two separate experiments) before continuing to PBMC isolation. Blood was collected during transfusion and kept in heparinized tubes to prevent clot formation. peripheral blood mononuclear cells (PBMCs) were isolated using 63% Percoll (Sigma, Cytiva;17-0891-01) solution, according to standard density gradient centrifugation methods (30 min, 400G). Samples were then treated with RBC cell lysis (Sigma, R7757, 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 10270106), Penicillin-Streptomycin-Amphotericin-B (Biological Industries, 1 03-033-1C)). In 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 (Trypsin 0.05%, no EDTA, Sartorius, 03-046-1B) 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, Using CG000204_ChromiumNextGEMSingleCell 3' v3.1_Rev D cDNA library preparation was were carried out according to the manufacturer's protocol.