GSM9065983: Untreated baseline, 10-week-old, pancreatic islet CD45+ immune cells; Mus musculus; RNA-Seq
The common bile duct was cannulated with a 27-G needle to infuse the pancreas with 3 mL of enzyme solution (HBSS with 25 mM HEPES, 2 mg/mL soybean trypsin inhibitor and 1U/mL Collagenase P). The inflated pancreas was removed and incubated at 37°C for 20 min with shaking every 2-3 min. Tissue digestion was stopped with culture medium (RPMI-1640 with 5% FCS, 2 mM L-glutamine, 100 U/mL penicillin + 100 μg/mL streptomycin, 25 mM HEPES) and the digested suspension was passed through a 450 µm mesh filter. Pancreatic islets were purified with density gradient centrifugation. Cell suspension was resuspended in 5 mL Lympholyte 1.1., overlayed with 5 mL of culture medium and centrifuged for 15 min at 24°C with brakes off. Enriched islets were collected from the Lympholyte/culture medium interface. To obtain single-cell suspensions, purified islets were digested with 1X StemPro Accutase at 37°C for 15 min with gentle vortexing every 2-3 min, washing with culture medium and passing through a 70 μm filter. Pancreatic islet-infiltrating immune cells were then FACS-purified as live CD45+ cells and resuspended in PBS with 5% FCS for downstream scRNA-seq library preparation. GEX libraries were constructed using 10X Genomics Chromium Single Cell 3' v3 chemistry with a target rate of 5k cells per sample according to the manufacturer's instructions by the University of Miami Sylvester Comprehensive Cancer Center Onco-Genomics Shared Resource core facility.
GEO
GSM9065983_r1