title: Illumina HiSeq 2500 sequencing; RNA-seq of human adipose-derived mesenchymal stem cells cultured in hypoxia against normoxia controls, and human tympanic membrane keratinocytes
design: RNA-seq of human adipose-derived mesenchymal stem cells cultured in hypoxia against normoxia controls, and human tympanic membrane keratinocytes
library_construction_protocol: After incubation, cells were rinsed in PBS twice before being lysed directly in FARB Buffer from the FavorPrepTM Tissue Total RNA Mini Kit (Favorgen Biotech Corp, Ping Tung, Taiwan) or Buffer RLT Plus from the RNeasy plus micro kit (Qiagen, Victoria, Australia). Human adipose derived mesenchymal stem cells were cultured in Dulbecco's Modified Eagle's medium (DMEM) containing 1 g/L D-glucose, 200 mM GlutaMAXTM and 110 mg/L Sodium Pyruvate (Gibco, Carlsbad, California), supplemented with 10% foetal bovine serum (FBS, Bovogen, Keilor East, Australia) and 1% Penicillin-Streptomycin (P/S; Gibco, Carlsbad, California). Spent medium was replaced every 2-3 days. Cells were incubated in standard culture conditions of 37oC, with 5% carbon dioxide (CO2) and atmospheric oxygen (O2) in a humidified cell culture incubator unless otherwise specified, and passaged 1:3 at approximately 80% confluence.  Human adipose derived mesenchymal stem cells were cultured to 80-90% confluence in 75cm2 tissue culture flasks, rinsed with 1x phosphate-buffered saline (PBS), pH 7.2 (Gibco, Carlsbad, California), and serum-starved for 8 hours before culturing with 10 mL fresh serum-free medium for a further 48 hours to produce conditioned media (CM). At this medium-conditioning step, cells were either incubated in standard conditions (Nx; 37oC, 5% CO2, ambient oxygen) or in hypoxic conditions (Hx; GENbox Jar; BioMérieux, France) produced by an anaerobic atmosphere generator sachet (BioMérieux, Craponne, France) at 37oC. A hypoxia indicator strip (BioMérieux, Craponne, France) was included in the sealed chamber to confirm gas composition (<0.1% O2 and 15% CO2). Total RNA was extracted using the FavorPrepTM Tissue Total RNA Mini Kit (Favorgen Biotech Corp, Ping Tung, Taiwan) according to the manufacturer's protocol, and cleaned up using the RNeasy Plus Micro kit (Qiagen, Victoria, Australia) according to the manufacturer's protocol. Quality of RNA was determined on the 2100 BioAnalyzer (Agilent Technologies, Victoria, Australia). All samples of at least an RNA Integrity Number (RIN) of ≥ 8.0 and an A260/280 ratio of 1.8 – 2.0 were accepted. Library preparation was performed on 1 µg total RNA per sample with the Illumina TruSeq® Stranded mRNA kit according to manufacturer's protocol (Illumina, San Diego, California).