GSM7848702: U3047-MG cells, Hypoxia 24h, replicate 2 [P15252_1032_S32]; Homo sapiens; RNA-Seq
Primary cells seeded into T25 flasks (0.3 - 0.5 x 106 cells/flask) and left to attach overnight (O/N) were pre-conditioned to 24 h normoxia/hypoxia (in triplicate experiments), detached with Accutase (Gibco, A1110501), collected in medium, and centrifuged for 5 min at 300 x g. The pellets were resuspended in phosphate-buffered saline (PBS) (Cytiva, SH30028.02) for cell counting, centrifuged for 5 min at 300 x g and each sample was resuspended in 350 µL RLT lysis buffer provided by the AllPrep DNA/RNA Mini Kit (Qiagen) supplemented with 1% (v/v) β-mercaptoethanol. Total RNA/DNA was then extracted as per manufacturer's instructions. Nucleotide concentration and purity were determined with NanoDrop (ND1000) and further confirmed with Agilent Bioanalyzer QC at the Bioinformatics and Expression Analysis (BEA) core facility (Karolinska Institute, Stockholm). RNA libraries for RNA-seq were prepared using the Trueseq stranded mRNA seq library kit as per the manufacturer's recommendations.