GSM5899196: R253_Low; Homo sapiens; RNA-Seq
After treatment incubation, all islets in 1 well were spun down at 300g for 3 minutes and supernatant was removed. The pellet was washed with magnesium and calcium-free PBS with 0.5mM EDTA. The cells were spun down once more and the PBS was removed. 50uL of PBS and 200uL of 0.25% trypsin/EDTA was added to the islets and they were incubated for 15 minutes at 37 degrees Celsius. The tube was mildly agitated with a flick every 2-3 minutes to disperse the islet cells. After trypsinization, 250uL of FBS and 1mL of PBS were added to neutralize the trypsin. The cells were spun down, the supernatant was removed, and the dispersed cells were resuspended in 300uL of PBS with 2% FBS for filtering through a 40um filter and counting on a hemacytometer. Once cell numbers were confirmed, the cells were spun down for a final time, the supernant was removed, and the cells were resuspended in a volume of magnesium and calcium-free PBS to achieve a single-cell solution of 1000 cells/uL. The resuspended cell solutions were placed on ice for immediate progression to single-cell library preparation. The 10x Genomics Chromium Controller was used to capture single cells, with each treatment condition from each donor islet preparation as 1 sample. cDNA libraries were prepared using 10x Genomics Single Cell 3' version 2 reagent kit and workflow for donors R253 and R282, and version 3 reagent kit and workflow for donor R317, according to the manufacturer's instructions.