Bulk transcriptomic analysis of primary beta cells from control, IGT, mild, and severely diabetic mice.
Mice were killed by cervical dislocation, pancreata were inflated with collagenase solution at 1 mg/ml, resected and placed in a water bath at 37°C for 10 min. After washing, islets were purified on a Histopaque-1119 / Histopaque-1083 gradient. Islets were cultured overnight at 37oC and 5% CO2 for respirometry, insulin secretion and enzyme studies, but were lysed ~3-4 hours after isolation for RNAseq analysis. Total RNA was isolated from islets using the ReliaPrep™ RNA Cell Miniprep System (Promega), according to the manufacturer's instructions. RNA quality was validated using an Agilent Bioanalyzer 2100. All samples had RIN values between 7.4 to 10.  500 ng of total RNA was used for library construction using Illumina's TruSeq Stranded mRNA prep kit following the manufacturer's instructions. Indexed libraries were purified and validated using an Agilent Bioanalyzer 2100. Sequencing libraries were pooled at equal molar concentration.