Illumina HiSeq 4000 paired end sequencing; RNA-Seq of pancreatic islets from whole body knockout of lncRNA Pax6os1 mice
RNA-Seq of pancreatic islets from whole body knockout of lncRNA Pax6os1 mice
Pancreatic islets (50-200) were extracted from female homozygous Pax6os1 knockout mice and their wild type littermate controls aged 22 weeks by pancreatic distension and digestion with collagenase. Islets were incubated in RPMI media containing 11 mM glucose, 10% FCS, 100 IU/mL penicillin, 100 Âµg/mL streptomycin, at 5% CO2 and 37 C for 24 h. RNA was extracted with TRIzol (Invitrogen) using the manufacturer's protocol, with the addition of glycogen as a co-precipitate. Resuspended RNA was treated with DNAse I (Invitrogen), which was then heat inactivated. Polyadenylated RNA was purified with oligo-dT beads and the library constructed using the TruSeq Stranded kit (Illumina). Illumina Universal Paired-end adapters were used, and custom barcoding sequences incorporated during the amplification rounds (15 cycles) for multiplexing.