title: GSM4829647: 1037R; Mus musculus; RNA-Seq
library_construction_protocol: OBs were lysed by the TRIzol Reagent.  Chloroform was added to samples and the tubes must be thoroughly vortexed. The tubes were centrifuged at 12,000 g for 15 min at 4°C. Upper aqueous phase was removed to a new tube and added with 250 μL isopropyl alcohol. After inverted a few times to mix, the samples were incubated for 10 min and centrifuged at 12,000 g for 10 min at 4°C. Supernatant was discarded by 1 mL pipet tip. After washing the RNA pellet with 950 μL 75% ethanol, the tubes were centrifuged at 7,500 g for 5 min at 4°C. The supernatant was discarded. The RNA pellet was dried and resuspended in RNase free water. RNA libraries were prepared for sequencing using standard Illumina protocols.Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesize dusing random oligonucleotides and SuperScript II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 200 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on a Hiseq platform (Illumina)