strain: 129 gender: male cell type: PGC-like sphere cells derived from ESC line #42 cell line: #42
Treatment protocol
4OHT, which conditionally activates the kinase activity of Akt-Mer fusion protein, was used at 1 microM in culture.
Growth protocol
The feeder-free mouse ES cells, E14tg2a and the derivatives were maintained as described (Niwa et al., 1998). The ES cells which expressed myr-Akt-Mer described in (Watanabe et al., 2006) were used. OP9 stromal cells were cultured in Minimum Essential Medium-Alpha (Gibco, Gaithersburg, MD, USA), supplemented with 20% fetal bovine serum (FBS) (JRH, 5M0161, Lenexa, KS, USA), 1% MEM non-essential amino acids solution (Gibco), 2.2 g/L sodium bicarbonate, 2 mM L-Glutamine (Gibco), 75 mg/L penicillin G and 50 mg/L streptomycin. The hematopoietic differentiation induction of ES cells on OP9 stromal cells has been described (Nakano et al., 1994; Nakano et al., 1996). Akt-spheres were produced by the same protocol as hematopoietic differentiation induction on OP9 cells in OP9 culture medium with 4OHT (Sigma-Aldrich, St Louis, MO, USA). 4OHT treatments of ES cells were started 3 days before the induction. Akt-spheres were expanded in OP9 culture medium with 4OHT on OP9 layer. Akt-spheres were passed onto freshly prepared OP9 cells after gentle pipetting or dissociation by trypsinization every 4 days and the medium was half changed every 2 days. The Akt-spheres were used for analysis after OP9 cells were removed by culturing on 90 mm dish (Nunc, Roskilde, Denmark) for 30 minutes. The MEFs were treated with 10 ug/mL mitomycin C in phosphate-buffered saline (PBS) for 2.5 hours and plated at 1.2×105 cells/well in 24 well plates or at 6×105 cells/well in 6 well plates.
Extracted molecule
total RNA
Extraction protocol
RNA samples were extracted from cells using RNesy mini kit (Quiagen).
Label
Cy5
Label protocol
Amino Allyl aRNA was synthesis by Amino Allyl MessageAmp II aRNA Amplification Kit (Ambion#1753). CyeDye Coupling and fragmentation were performed as the protocol supplied by TORAY Industries, Inc..
Hybridization protocol
Hybridized for 16 h at 37 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc..
Scan protocol
ScanArray Express (PerkinElmer Japan Co., Ltd.) was used for scanning. Images were quantified using GenePix Pro version 6.0(Axon Instruments).
Description
Akt-spheres derived from ESC#42 +4OHT
Data processing
F635 Median column as Red signal. Global normalized, background subtracted data obtained from log2 of processed Red signal/processed Red signal.
