Illumina HiSeq 2000 sequencing; Transcription profiling by high throughput sequencing of pancreatic islets from LKB1 and AMPK knockout mice
Transcription profiling by high throughput sequencing of pancreatic islets from LKB1 and AMPK knockout mice
Pancreatic islets (50-100) were extracted from Ins1LKB1KO mice or Ins1AMPKdKO and their wild type controls aging 12-14 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 ug/mL streptomycin, at 5% CO2 and 37 C for 24 h. RNA was extracted using RNAeasy kit according to the manufacturer's instructions (Qiagen) All libraries were prepared using the TruSeq RNA Sample Preparation Kits v2 (Illumina, San Diego, CA USA) according to the manufacturer's instructions. In brief, magnetic beads containing poly-dT molecules were first used to purify mRNA from 250 ng of total RNA. Second, samples were chemically fragmented and reverse transcribed into cDNA. Finally, end repair and A-base tailing was performed before Illumina adapters were ligated to the cDNA fragments. Purified samples were amplified by 15-cycle PCR. Amplified material was validated and quantified using an Agilent 2100 bioanalyzer and the DNA 1000 Nano Chip Kit (Agilent, Technologies, Santa Clara, CA, USA).
Experimental Factor: genotype
floxed AMPK without RIP-Cre
Experimental Factor: phenotype
wild type