title: GSM4928856: pancreatic islets, FACS-sorted for mTomato and mGFP fluorescence; Mus musculus; RNA-Seq
library_construction_protocol: Islets of Langerhans were isolated from mice. Mice were sacrificed by cervical dislocation. The abdominal cavity was  opened and pancreas, common bile duct and gallbladder exposed. The ampulla was sealed with a bulldog clamp and Collagenase P (Roche) (1 mg/mL in G-solution) injected in the bile duct with a syringe and a 30G needle. Once the organ was perfused with 3ml of the abovementioned solution, pancreats was dissected and incubated in 3ml of Collagenase P solution and placed on ice. Afterwards, the pancreas was digested at 37°C for 12 minutes and shaken vigorously at least once at half the time of incubation. The digestion was stopped on ice by adding 10ml G-solution plus 10% FBS. The mixture was centrifugated (1620rpm, 3 min, 4°C) in order to pellet the digested tissue and remove the solution containing the collagenase. The pellet was then resuspended in 5.5ml of gradient solution (40%Optiprep). This was then centrifugated (1700rpm, 10 min, RT, acceleration 3 and deceleration 0). Islets were sedimented at the interface between the first and second phase. They were collected and washed on a 70µm cell strainer with G-solution, then placed in a 10cm dish in islets medium. Considering that the obtained solution is still rich in exocrine cells, islets were handpicked and placed in a new dish with fresh medium. Islets are cultured at 37°C and 5%CO2. Islets were then picked in an 1.5 mL Eppendorf tube, pelleted (800 rpm, 2 min) washed with PBS (-Mg/Ca) and digested with TrypLE™ Express Enzyme (1X) (Thermo Fisher Scientific) at 37 °C for 10 min, gently pipetting every 2 minutes. After this, the tube was placed in ice and FACS buffer was added to the cells and finally centrifuged at 900rpm for 5 min. The supernatant was discarded and an appropriate amount of FACS buffer was added to resuspend the single cells. Finally cells were sorted in the FACS Aria III into 3 sepaerate tubes.
attributes.tag: GEO Accession
attributes.value: GSM4928856