For reaggregation cultures to support the differentiation of the hematopoietic and vascular lineages, 3 x 10^5 Flk-1+ cells/ml (harvested at d3.5 after addition of serum) were cultured for 24 hours in ultra-low attachment 24-well plates (Corning Costar) with the EB differentiation medium (above) plus 5% Protein-Free Hybridoma Medium-II (PFHM-II, Invitrogen), either in the presence or absence of dox. After 24 hours, the cells were washed and cultured without doxycycline for up to an additional 3 days. Total RNA was harvested from dox-treated or dox-null cultures at 4, 12, 24, 48, or 96 hours.
Growth protocol
ES cells were maintained on irradiated feeders in Dulbecco€'s Modified Eagle Medium (DMEM) supplemented with 15% fetal calf serum (FCS), 10% ES cell conditioned medium, penicillin, streptomycin, 1.5 x 10^-4 M monothioglycerol (MTG; Sigma) and LIF (1% conditioned medium). Prior to induction of differentiation, cells were passaged 2 times on gelatin-coated plates in Iscove Modified Dulbecco Medium (IMDM) containing the same supplements mentioned above to deplete the population of feeder cells. ). Prior to induction of differentiation, cells were passaged 2 times on gelatin-coated plates in Iscove Modified Dulbecco Medium (IMDM) containing the same supplements mentioned above to deplete the population of feeder cells. For the generation of EBs, the cells were harvested and cultured in 60 mm low attachment Petri grade dishes (VWR) with IMDM supplemented with 2 mM L-glutamine (Gibco/BRL), 200 µg/mL transferrin (Boehringer Mannheim), 0.5 mM ascorbic acid (Sigma), 4 x 10^-4 M MTG plus 15% FCS.
Extracted molecule
total RNA
Extraction protocol
Qiagen RNeasy Mini protocol for isolation of total RNA from animal cells, with on column Dnase treatment.
Label
Alexa-647
Label protocol
see CodeLink Gene Expression System: Manual Labelled cRNA target Preparation
Hybridization protocol
see CodeLink Gene Expression System: Single-Assay Bioarray Hybridization and Detection
Scan protocol
see CodeLink Gene Expression System: Single-Assay Bioarray Hybridization and Detection, using Axon 4000B scanner
Description
***** These records currently present results containing only probes for the Wnt, BMP/TFGb, and Notch pathways as published in Chen et al., 2008, Nature Biotechnology. The full set of processed and raw data tables will be available by June 1, 2009. *****
Data processing
median normalization, CodeLink Expression Analysis, filtered for Wnt, BMP/TFGb, and Notch pathway probesets
