hypoxia replicate3
Caki-1 human renal carcinoma cells were cultured in DMEM supplemented with 10% FBS. For hypoxia treatment, we placed cells into a CO2 incubator with O2 control (Binder) with regulated environment of 1%O2, 5% CO2 and 94% N2, or cobalt chloride (CoCl2) 300 mkM (stock solution 100mM in water) for 24 h. Total RNA was extracted from Caki-1 cells with Trisol reagent according to manufacture instruction (Invitrogen). Quality was checked with  BioAnalyser and  RNA 6000 Nano Kit (Agilent). PolyA RNA was purified with Dynabeads® mRNA Purification Kit (Ambion).  Illumina library was made from polyA RNA with NEBNext® mRNA Library Prep Reagent Set (NEB) according to manual. Sequencing was performed on HiSeq1500 with 50 bp read length. 10 millions of reads were generated for each sample.