title: Dynamic Ca2+-dependent transcription links metabolic stress to impaired β-cell identity
design: Dynamic Ca2+-dependent transcription links metabolic stress to impaired β-cell identity
library_construction_protocol: Male mice were used for islet isolations at 8-9 weeks of age. Mouse islets were isolated following an injection of 0.6 mg/mL Collagenase P (Roche) into the pancreatic bile duct. Partially dissociated tissue was fractionated using a Histopaque-1077 (Sigma) gradient followed by hand-picking of islets. Islets were incubated in vitro following the treatment protocol and then used for RNA isolations. C57BL/6J mice fed regular chow (4.5% fat content, PicoLab, 5L0D) were used for all experiments. Ascl1β knockout (KO) mice were obtained by interbreeding animals with the Ascl1tm2Fgu, Ins1tm1.1(cre)Thor and Gt(ROSA)26Sortm9(CAG-tdTomato)Hze alleles. Mouse islets were isolated at 8-9 weeks of age. Isolated islets were cultured overnight (16 hours) in islet culture media consisting of low (1 mg/L) glucose DMEM (Thermo, 11966-025) supplemented with 10% FBS (Atlanta Biologicals), 4.6 mM HEPES, 1% penicillin/streptomycin (Thermo), 1% non-essential amino acids (Sigma) in 24-well plates at 37°C with 5% CO2 infusion and 95% humidity. For time series experiments, wild type islets were treated with culture media containing 100 μM tolbutamide (Sigma, T0891) for 0 hours (control), 0.5 hours, 2 hours, 6 hours, and 24 hours and then collected for RNA isolations.   For combinatorial treatment experiments, islets were treated for 24 hours as following: 1) with 100 μM tolbutamide (Sigma, T0891); 2) with combination of 100 μM tolbutamide and 50 μM verapamil (Sigma, V4629); 3) with combination of 100 μM tolbutamide (Sigma, T0891) and 1 μM CREB-inhibitor 666-15 (MedChemExpress, HY-101120); with high glucose (20 mM),  Control samples were treated only with the DMSO vehicle (0.1%). Islets from Ascl1βKO mice were treated for 24 hours with tolbutamide. Total RNA was extracted from isolated mouse pancreatic islets using Maxwell simplyRNA kits (Promega). cDNA libraries were constructed using low input Illumina library prep workflow at Novogene.