GSM9039693: KPC-IMPACT-OVEREXPRESSION - GEX; Mus musculus; RNA-Seq
Liver extirpated from a euthanized mouse was disrupted through a 0.75um cell strainer using the rubber side of a 3cc syringe into a 50ml tube with 5-10ml of RPMI media for each liver/lung. The media containing cells from the liver/lung was centrifuged at 30g for 3 minutes to remove the hepatocytes and tumor cells, and the supernatant was transferred to a new tube. Following centrifugation at 1500 rpm for 5 mins, the cell pellet was resuspended in 32% Percoll solution and again centrifuged at 140C, 2000 rpm with no brake for 15 mins. RBCs were removed by washing the cell pellet with 5 ml of ACK lysis buffer and centrifuging the samples at 1500 rpm for 5 mins. The cell pellet containing only immune cells was further washed 2X in complete RPMI media and used for surface and intracellular staining with specific antibodies. Single-cell suspensions were prepared and cell counts and viability measurements were taken for each suspension sample using a fluorescent cell counter with dual viability dyes composed of propidium iodide and acridine orange (Luna-FL, Logos Biosystems). Sample concentrations were adjusted and loaded onto the 10x Genomics Chromium platform using the 10x Genomics 3' v4 Gene Expression Assay, targeting roughly 20,000 cells per sample. Preparation of single-cell gene expression libraries was performed according to the vendor's recommendations.