title: GSM2805318: Subc. adipose tissue Type 2 diabetes patient-6 at ZT D2:1530; Homo sapiens; RNA-Seq
library_construction_protocol: RNA isolation was performed according to the RNeasy-Mini protocol for animal tissues and cells on the Qiacube (Qiagen) including an RNAse-free-DNase digestion step. RNA concentrations were measured with a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), and quality was checked on a RNA 6000 Nanochip with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Subsequently cDNA was constructed with the Ovation RNA-Seq System V2 (NuGen, Manchester, UK) and the product was purified with the MinElute PCR Purification kit (Qiagen). cDNAconcentration was determined by fluormetric measurement with Qubit dsDNA BR (Invitrogen, Thermo Fisher Scientific) and quality checked with the Bioanalyser  DNA1000 (Agilent Technologies). 2 ug cDNA was sheared by sonication (Covaris, Woburn, MA, USA)  using the following settings: 7 cycles, frequency sweeping, 10% duty cycle, intensity 5, 100 cycles/burst, 60 seconds. Size distribution after shearing was checked using the Bioanalyser. Sheared cDNA was size selected to maintain fragments in the size range of 140 to 180 bp, by double ampure purification (Agencourt, Beckman Coulter, Brea, CA, USA) on a Biomek FX automated liquid handler (Beckman Coulter) according to the manufacturer’s recommendations, an ampure:sample ratio of 1.0 for the first bead capture, and a ratio of 1.5 for the second bead capture. Sizing and concentration were checked using Bioanalyser and Qubit. Barcoded adaptor ligated library construction, from the size selected cDNA, was performed using the Biomek FX automated liquid handler, and the Solid fragment Library preparation kit (Lifetech, pn 4464412), and Solid barcoded adaptors following manufacturer’s protocol. Each sample was generated using a separate 10bp barcode incorporated in the adaptor sequence. After 8 cycles of amplification using the library prep kit supplied PCR primers, PCR mix, and PCR protocol, samples were purified twice using ampure (ratio of 1.5) and pooled equimolar based on concentration and average sample size. Sequencing of this equimolar pool was performed on the Solid 5500 Wildfire sequencer (Life technologies, Thermo Fisher Scientific) generating an average of 40 million 50bp fragment and 10bp barcode reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM2805318