title: Illumina HiSeq 2000 sequencing; Human primary adipose-derived stem cells (hpASC) differentiated to white adipocytes stimulated with norepinephrine 3h
design: Human primary adipose-derived stem cells (hpASC) differentiated to white adipocytes stimulated with norepinephrine 3h
library_name: hpASC1_VE_s
library_construction_protocol: Cells were harvested with TRIzol reagent (Invitrogen) 3 h after stimulation with NE. Human primary adipose-derived stem cells (hpASCs) were isolated from subcutaneous lipoaspirates from healthy female donors (n=4). Cells at P0 were thawed, cultivated in EGM-2 Medium (Lonza), and used after 1-3 passages. For adipocyte differentiation, cells were seeded in 6-well plates (85000 cells / well) in EGM-2 Medium. Cells reached 100% confluence after 3 days, when the medium was replaced with fresh EGM-2 Medium. 2 days later (= day 0), adipocyte differentiation was induced by changing the medium to adipocyte differentiation (AD) medium (DMEM/Ham's F12 (50:50), 5 mM HEPES, 2 mM L-glutamine, 100 µg/ml normocin, 860 nM insulin, 10 µg/ml apo-transferrin, 100 nM rosiglitazone, 0.2 nM triiodothyronin) supplemented with 100 µM 3-isobutyl-1-methylxanthine (IBMX), and 1 µM dexamethasone (Dex). Medium was replaced at days 2 and 5 with AD medium. At day 7, another medium change was performed using AD medium without insulin. Adipogenesis was monitored and confirmed based on the appearance of lipid droplets by microscopy At day 9 after differentiation, norepinephrine (NE) stimulation was performed by changing the medium to AD medium without insulin, but supplemented with 25 µg/mL ascorbate and either 1 µM norepinephrine (NE; dissolved in 10 mM HCl) or vehicle (VE, 10 mM HCl).  RNA isolation was performed with TRIzol reagent (Invitrogen), according to the manufacturer's protocol. Quality of RNA was assayed by an RNA Nano chip using the BioAnalyzer 2100 (Agilent); all samples had RIN values ≥8.5 4 µg total RNA per sample were used with the TruSeq Stranded mRNA LT Sample Prep Kit (Illumina) to generate cDNA libraries according to the manufacturer's protocol.
attributes.tag: Experimental Factor: stimulus
attributes.value: none