GSM8408128: Sheep pancreas CF Term scRNAseq donor 3B scRNA-seq; Ovis aries; RNA-Seq
For single cell isolation from fresh pancreas, ~ 300 mg of tissue was cut into 2-3 mm pieces and subjected to two rounds of digestion: First in 5ml of collagenase solution (200 U/ml collagenase, 40 IU/mL, DNAse I 250 μg/mL, soybean trypsin inhibitor (SBTI) (all from Worthington), 3 mM CaCl2, at 37°C, with continuous stirring for 30-40 min. Tissue clumps were then disassociated by pipetting 10 times in a 5 ml serological pipette and left to settle for 3-4 minutes at which point the supernatant was collected and stored on ice as cell suspension 1. Second, 5 ml of fresh collagenase digestion solution as above was added to the settled tissue clumps remaining after the first digestion and again stirred at 37°C for 30-40 min, at which point very little tissue debris remained. The supernatant (cell suspension 2) was pooled with cell suspension 1 and together passed through a cell strainer (200 µm, pluriSelect) to remove any tissue debris. The strained cell suspension was then centrifuged at 300 ×g for 5 min, washed once in HBSS (Sigma Aldrich) + 2% FBS and centrifuged again. 4 ml of 1× Red Blood Cell Lysis Solution (Miltenyi Biotec 130-094-183) was added to the cell pellet which was vortexed for 5 seconds and then incubated for 5 minutes at room temperature. After 10 mL of HBSS with 2% FBS was added, the mixture was centrifuged at 300 ×g for 5 min, and the supernatant was removed. The cell pellet was then digested with 3 mL of Accutase (Stem Cell Technologies) for 15-25 min with frequent gentle pipetting with a wide-bore pipette, after which time the cells were visualized under a microscope. If any cell clumps persisted, they were collected by centrifugation and further digested with replace with 3 mL of Accutase, and digested for 10-15 min. Accutase was removed by washing the cells with 5 mL HBSS + 2% FBS, followed by centrifugation to collect the cells. The supernatant was discarded, and the cell pellet was resuspended in 0.5-1.0 ml HBSS, passed through a 20-µm mini cell strainer (pluriSelect) and cells counted. ~ 2000 - 6,000 cells were used for scRNA-seq using the 10x Genomics Chromium system. Libraries generated from single cells according to the manufacturer's protocol for 10X Genomics Chromium Single Cell 3' Reagent Kits v3 or 3.1.