GATA2 mutant transduction of murine hematopoietic stem cells

GATA2 mutant transduction of murine hematopoietic stem cells C57BL/6 8-week female were treated with 5-fluorouracil (5-FU) at 150mg/kg 6 days prior to bone marrow cell harvesting. The bone marrow cells were infected with MIE, WT and R396Q retroviral particles. GFP-positive cells were sorted after 72h of culture. Total RNA was extracted using Rneasy plus MiniKit (Qiagen) and its quality was assessed with the RNA Screen Tape on Agilent 2200 Tapestation Bioanalyzer (Agilent Technologies). RNA samples were purified and prepared according to the manufacturer's protocol using the Affymetrix's GeneChip Whole Transcript Sense Target Labeling Assay Kit (Affymetrix) and hybridized on Affymetrix GeneChip Mouse Exon 1.0 ST arrays. Probe-signal intensities were normalized and summarized using the robust multiarray average (RMA) method with the Affymetrix’s Expression Console software. 3 controls (MIE), 3 GATA2 wild type, 3 GATA2 R396Q