GSM1462750: CAST; Mus musculus; RNA-Seq
Male C57BL/6J (B6) and CAST/EiJ (CAST) mice were purchased from The Jackson Laboratories (Bar Harbor, Maine) and housed in an environmentally controlled vivarium at the University of Wisconsin Biochemistry Department. Mice were provided standard rodent chow (Purina no. 5008) and water ad libitum, and maintained on a 12-hour light/dark cycle (6 AM – 6 PM). At 10 weeks of age, mice were sacrificed by CO2 asphyxiation. All animal procedures were preapproved by the University of Wisconsin Animal Care and Use Committee. Intact pancreatic islets were isolated using a collagenase digestion procedure as previously described.[20] Briefly, 5 ml of collagenase type XI (Sigma), dissolved in Hanks’ balanced salt solution (Gibco), was injected into the pancreas via the common bile duct (0.45 mg/ml with 0.02% BSA). The pancreas was removed and incubated at 37°C for 16 min with intermittent agitation. A ficol gradient was used to partially purify islets from the digested pancreas, followed by further purification by manual picking under a stereomicroscope. During the purification procedure, islets were maintained in Krebs-Ringer bicarbonate buffer containing (mM): 118.41 NaCl, 4.69 KCl, 2.52 CaCl2, 1.18 MgSO4, 1.18 KH2PO4, 25 NaHCO3 and 5 HEPES supplemented with 0.2% BSA and 16.7 mM glucose. Islets from two B6 mice (and separately two CAST mice) were pooled and then apportioned for RNA-Seq (250 islets per sample) and protein analyses (remainder: 400 B6 islets, 470 CAST islets). For RNA profiling, whole islet RNA was purified as previously described yielding ~1 µg total RNA.[21] For protein analyses, islets were washed 3 times in ice-cold PBS, gently pelleted and snap frozen in liquid nitrogen.