title: GSM6204247: CD63 low, replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: Mouse pancreatic islets from 10 weeks-old C57BL/6J male mice were isolated by perfusion of the pancreases with CIzyme (VitaCyte) through the common hepatic duct and digested at 37°C for 17 minutes. After gradient-based separation using Histopaque-1077 (Sigma), islets were then hand-picked to avoid exocrine contamination. Pancreatic islets were isolated and cultured overnight in RPMI 1640 supplemented with 10% FBS and penicillin/streptomycin. Then, islets were dissociated with 0.05% Trypsin (Corning) for 10 minutes at 37°C. Dissociated cells were passed through a 35 μm nylon mesh and resuspended in 1x PBS + 0.05% BSA. Then, we FACS-sorted beta cells based on CD63 expression. After RNA isolation, total RNA integrity was analyzed using an Agilent 2100 Bioanalyzer and concentrations were measured using the NanoDrop Spectrophotometer. Preparation of the RNA sample library were performed by the Genomics Core Laboratory at Weill Cornell Medicine. cDNA was generated using the SMARTer v4 Ultra Low Input RNA Kit (Clontech, # 63488) and Nextera XT kit (Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA) was used to make cDNA libraries suitable for Illumina sequencing.