GSM6215268
As each region was sampled, the tissue was chopped into 1mm x1mm cubes and placed in 0.25mg/ml liberase TH (Roche Diagnostics, Indianapolis, USA) dissociation medium. Following further dissection, the samples were incubated at 37°C for 1 hour at 600rpm. Samples were regularly triturated during the incubation period using a 1ml pipette, after which 10% heat- inactivated FBS RPMI was added to stop the digestion. Centrifugation at 500 g for 5 minutes at room temperature with the removal of the supernatant, was followed by the addition of ACK lysing buffer to remove erythrocytes (Thermo Fisher Scientific, Waltham, USA). Following centrifugation, the resulting cell pellet was incubated with Accumax at 37°C for 3 minutes (Innovative Cell Technologies Inc, San Diego, USA). 10% FBS RPMI was again used to neutralize the Accumax and centrifugation was followed by resuspension of the cell pellet in 0.4% BSA/PBS. The single cell suspension was filtered using a 30um CellTrics filter (Sysmex America Inc, Lincolnshire, USA) with cell viability and concentration determined using trypan blue and the Auto T4 cellometer (Nexcelom Bioscience LLC, Lawrence, USA). 17,500 cells were loaded into the 10x Genomics microfluidic system/onto the 10x Genomics platform to achieve a targeted recovery of 10,000 cells (10x Genomics, Pleasanton, USA).