title: GSM6249853: OB-TX-2; Mus musculus; RNA-Seq
library_construction_protocol: OBs (two for each mouse, from 3 mice each condition) were collected and homogenized in 500 mL TRIzol reagent (ThermoFisher). Samples were incubated for five minutes at 4°C and 100 mL chloroform was subsequently added. Samples were centrifuged for fifteen minutes at 12,000 x g at 4°C and the aqueous layer was collected and transferred to a 1.5 mL centrifuge tube. 250 mL of isopropanol was added to samples and incubated for ten minutes at 4°C. The samples were centrifuged for ten minutes at 12,000 x g at 4°C and the supernatant discarded. Pellets were resuspended in 500 mL 75% ethanol and vortexed before being centrifuged for five minutes at 7,500 x g at 4°C. Pellets were allowed to dry and rehydrated in 35 mL of RNase free ultrapure water. Samples were incubated at 60℃ for fifteen minutes to complete RNA isolation. RNA concentrations and purity were assessed using a NanoDrop Lite Spectrophotometer (Thermo Scientific). Sample quantity and RIN values were determined. Libraries were prepared using NEBNext Ultra RNA Library Preparation Kit according to the manufacturer's protocol (New England Biolabs).