GSM5461049: KO-5; Mus musculus; RNA-Seq
Control (TNFR1+/-) and TNFR1-/- primary CMFs were seeded at a density of 1x103 cells/50µL of Matrigel into 24-well plates and were grown to 70% confluency. At Day 5, CMFs were extracted from Matrigel after brief incubation at 4⁰C via centrifugation at 1200rpm for 5 min. Cells were reconstituted in 10mL of HBS/1% BSA solution and were passsed through a 40 µm filter to suspend into single cells. To remove dead cells for the final flow, single cell suspensions of control of TNFR1-/- CMFs were stained with the EasySep™ Dead Cell Removal (Annexin V) Kit (Thermofisher) according to manufacturer's guidelines. Live cells were quantified and reconstituted in 100-200 µL of HBS/1% BSA solution. Samples with live cell counts of 95% and above only were processed for scRNA-Seq. Single-cell 3' libraries were generated using the 10X Chromium platform. Paired-end sequencing with index reads was performed on a NextSeq 500.