GSM9312989: non-CD4 T cells 2 GEX; Mus musculus; RNA-Seq
Islet infiltrating cells were isolated from four Cre+-Tnfrsf9+/+ and four Cre+-Tnfrsf9fl/fl 7-10-week-old females The pancreas was inflated using a 30-gauge needle by injecting 3-5 mL of HBSS containing 0.5 U/mL collagenase P solution (Roche Diagnostics) and 10 μg/mL DNase (Sigma) into the bile duct. The inflated pancreas was digested at 37°C for 15 min. Next, the digested pancreas was washed three times with 10 mL of HBSS containing 2% FBS. Each individual pancreas was then suspended in 5 mL complete RPMI 1640. Islets were visualized under a dissecting microscope and hand-picked. Pelleted islets were suspended in 200 μL of enzyme free cell dissociation buffer (Life Technologies). Cells were then washed with 500 μL of HBSS, resuspended in FACS buffer. Cells were incubated with 5 µg/mL Fc block (anti-mouse CD16/CD32 clone 2.4G2, BioXCell). Each cell pool was then stained with anti-CD45.1, anti-CD4, and a unique hashtag antibody for 30 minutes at 4ºC, washed, and passed through a 30 µm filter. All stained cells were combined and simultaneously sorted into CD45.1+CD4+ and CD45.1+CD4- samples on a FACSAria III (BD Biosciences). Sequencing libraries were prepared using the Chromium Next GEM Single Cell 5' Reagent Kits v2 (10X Genomics).