GSM5655259: KO_rep1; Mus musculus; RNA-Seq
Isolated mouse islets were dispersed in trypsin (0.05%) for 5 minutes at 37⁰C. To image proliferative β-cells, dispersed islets were seeded in chamber slides (ThermoFisher Scientific) coated with poly-D-lysine hydrobromide (Sigma-Aldrich), and cells were cultured in RPMI1640 medium containing 11 mM glucose with 10% FBS for 72 h in the presence of 10μM of BVO2, 14-3-3i and harmine (Sigma-Aldrich). Media were changed every 24 h. After successive washes with PBS, cells were fixed with 4% paraformaldehyde, followed by co-immunostaining for insulin (ab63820;1:50 dilution; Abcam) and the proliferative markers Ki67, (ab15580; 1:300; Abcam). Single-cell RNA-seq libraries were made with the 10x Genomics Chromium Next GEM Single Cell 3' Library Kit (v3.1) and sequenced using an Illumina NovaSeq6000 (100bp x2, 50k reads/cell). Approximately 9600 cells were loaded per sample with an anticipated recovery of 6000 cells.