title: AHC4
organism: Mus musculus
description: Protocols: Islets from male mice were isolated at 14-17 weeks of age by injecting 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 from individual mice were used for separate RNA sample isolations. All mutant alleles were bred into a C57Black/6J genetic background.  Ascl1Flox (Ascl1tm2Fgu), InsCre (Ins1tm1.1(cre)Thor) and Rosa26tdTomato (Gt(ROSA)26Sortm9 (CAG-tdTomato)Hze) alleles were interbred to create the beta-cell specific Ascl1 knockout (Ascl1betaKO).   Ascl1betaKO and control heterozygous islets were used in the experiment. Mice were either fed regular chow (RC) (4.5% fat content, PicoLab, 5L0D) or HFD (60% fat content, D12492, Research Diets) for 12 weeks.  Ascl1betaKO mice were also bred into homozygous Abcc8 KO (Abcc8tm1.1Mgn) mouse background.   Total RNA was extracted from isolated mouse pancreatic islets using the Maxwell simplyRNA kit (Promega). cDNA libraries were constructed using Illumina library prep kit at Novogene.