GSM4852142: Islet preparation 7 under control condition at D4; Homo sapiens; RNA-Seq
RNA from approximately 120 hand-picked islets per condition was prepared using the RNeasy MINI Kit + QIAshredder (Qiagen, Hilden, Germany) through lysing and homogenizing steps followed by DNA digestion using RNase-Free DNase Set (Qiagen) and washing steps. Total RNA concentration was measured using the NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA) and RNA RIN values were obtained using Agilent Bioanalyzer 2100 (Agilent Technologies, Wokingham, UK) and Agilent RNA Nano Chips (Agilent Technologies). RIN values in the ND samples were: mean 8.9; SEM 0.1; median 8.8, range 7.9–9.7. The samples used for the T2D vs ND islet comparison had a RIN value of 7.5 and above, ensuring suitability for sequencing. Libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). Briefly, mRNA was purified from 0.40 µg total RNA using poly-T oligo attached magnetic beads, and was then fragmented and primed with random hexamers for reverse transcription. Afterwards, second strand synthesis was performed by incorporating dUTP in place of dTTP to generate blunt-ended ds cDNA. Adenylation of the 3' ends and adaptor ligation were then performed. Selective enrichment of DNA fragments with adapter molecules on both ends and DNA amplification were obtained by PCR. The generated libraries were quantified using qPCR and quality was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies) and Agilent DNA 1000 chips (Agilent Technologies).