cell type: EB cells cultured with a hematopietic cytokine cocktail for 10 days
Growth protocol
To differentiate ES cells into HSCs, a 2-stage culture strategy was adopted. Initially, ES cells were subjected to embryoid body (EB) formation over 6 days. At this stage, ES cells were plated to an ultra-low attachment Petri dish at a concentration of 2000 cells/mL in a methycellulose-based differentiation medium containing Iscove modified Dulbecco medium (Invitrogen), 15% FCS, 300 μg/mL transferrin, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 5% protein-free hybridoma medium (Invitrogen), 4x10-4 M monothioglycerol, and 50 μg/mL ascorbic acid. At second stage, EBs were dissociated into single-cell suspension using trypsin (2.75%), and 3.5x106 cells/mL cells were re-plated on another ultra-low attachment Petri dish in a serum-free hematopoietic differentiation medium that contained StemPro34 plus nutrient supplement (Invitrogen) and a cocktail of hematopoietic cytokines including murine stem cell factor (mSCF; 100 ng/mL; R&D Systems, Minneapolis, MN), mIL-3 (2 ng/mL), mIL-6 (5 ng/mL), Flt3-L (10 ng/mL), insulin-like growth factor-1 (IGF-1; 40 ng/mL; Promega, Madison, WI), and dexamethasone (1 μM; Sigma-Aldrich, St Louis, MO). Culture medium was changed every other day and cell density maintained below 4x106 cells/mL.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from cultured cells using Trizol reagent according to the manufacturer's protocol (Invitrogen). RNA samples were treated with Dnase I to remove genomic DNA contamination. RNA quality was assessed by its absorbance at 260nm and 280nm using a NanoDrop spectrophotometer and its integrity was assessed bioanalyzer.
Label
biotin
Label protocol
Total RNA underwent first-strand and second strand cDNA synthesis. Complementary RNA was generated and used to produce sense-strand cDNA, which was fragmented and end-labeled with biotin. Microarrays were hybridized, washed, stained, and scanned according to the protocol described in the WT sense target labeling assay manual from Affymetrix.
Hybridization protocol
according to the instruction manual, as described in the Affymetrix website.
Scan protocol
according to the instruction manual, as described in the Affymetrix website.
Description
day_16_rep_2
Data processing
Microarray data were normalized using the RMA algorithm.
