Single cell RNAsequencing from cultured omental adipose tissue derived preadipocytes.

Preadipocytes derived from visceral (mental) adipose tissue stromal vascular fraction (SVF) cultures from four diabetic patients were cultured in vitro until confluency. Single-cell suspensions were obtained by detaching preadipocytes with 5 mL of 0.25% trypsin (ThermoFisher Scientific Inc./Gibco) for 3 min at 37C, followed by centrifugation (500 rcf, 5 min, 4C), and resuspension with 1mL of 1X DPBS with 0.04% BSA with gentle mixing using a wide-bore tip. A pre-wet 40 um cell strainer was used to remove any remaining cell debris and large clumps. A 10 uL sub-sample was obtained to determine viability and the cell concentration using trypan blue and automated cell counter. All samples had cell viability >95%. Cell suspensions were centrifuged at 300 rcf for 5 min, supernatant removed, and single-cell suspensions diluted to 1000 cells/uL using 1X DPBS with 0.04% BSA. Cells were used to generate single cell libraries using the 10X genomics platform.