GSM8973672: Wilms tumor_CMCW17; Homo sapiens; RNA-Seq
Nuclei were isolated as previously reported from paired kidneys with Nuclei EZ Lysis buffer supplemented with protease inhibitor and RNase inhibitor. Samples were homogenized using a Dounce homogenizer in 2ml of ice-cold Nuclei EZ Lysis buffer first with a loose pestle, then passed through a 200 µm strainer into 50 ml conical tube and followed by homogenization with a tight pestle which was then filtered through a 40-µm cell strainer and incubated on ice for 5 minutes. The suspension was centrifuged at 500 g x for 5 min at 4 ºC with the pellet resuspended and washed with 4 ml of the buffer and incubated on ice for 5 min. After another centrifugation, the pellet was resuspended with Nuclei Suspension Buffer (1x PBS, 0.07% BSA, 0.1% RNase inhibitor) and filtered through a 5-µm cell strainer into a conical tube. The nuclei were counted with a disposable hemacytometer and diluted to a concentration of 700-1200 cells per µL. Library was performed according to the manufacturer's instructions (single cell 3' GEX kit).