title: GSM9231907: B3 Control; Homo sapiens; RNA-Seq
library_construction_protocol: Each shRNA was expressed in one lentiviral DNA construct under the control of a U6 promoter, which also expresses eGFP for monitoring transfection. Islets were dissociated into single cells, washed with standard CMRL islet media (CMRL1066 with 10% heat-inactivated FBS, 1X antibiotics, and 1X L-glutamine) and resuspended in VPM media (50% CMRL1066 + 50% Vasculife Basal Media, 10% heat-inactivated FBS, 1X antibiotics, 0.5X Glutamax, 2.5 mM HEPES, 0.5X sodium pyruvate plus rh VEGF LifeFactor, rh EGF LifeFactor, rh FGF basic LifeFactor, rh IGF-1 LifeFactor, Ascorbic Acid LifeFactor, Hydrocortisone Hemisuccinate LifeFactor, Heparin Sulfate LifeFactor, Gentamicin/Amphotericin, L-glutamine LifeFactor B, and iCell Endothelial Cells Medium Supplement). Cells (200,000 in 200 μL) were aliquoted into one prepared Aggrewells 96-well plate, mixed with 40-50 ng of lentivirus. The plate was centrifuged at 200g for 5 min and left undisturbed in a cell culture incubator for 5 days. The PSIs were then recovered for downstream studies. Wells preparation uses 100ul/well of anti-adhesive rinsing solution (96-well late, Nacali #4860-900SP). dissociated PSIs with ~80-90% single cells and <5% dead cells were used for InDrop. The end library was sequenced with Novaseq 6000 (Illumina), targeting 120 million reads per sample.