age: postnatal day 3 tissue: fore and hind limbs genotype: wild-type cell population: CD45-Ter119-Tie2-AlphaV+Thy-6C3+CD105-
Growth protocol
Skeletal tissues were dissected from P3 GFP-labeled mice and dissociated by mechanical and enzymatic dissociation. Specifically, the tissue was placed in collagenase digestion buffer supplemented with DNase and incubated at 37oC for 40 minutes under constant agitation. After collagenase digestion and neutralization, undigested materials were gently triturated by repeated pipetting. Total dissociated cells were filtered through 40 m nylon mesh, pelleted at 200g at 4oC, resuspended in staining media (2% fetal calf serum in PBS), blocked with rat IgG and stained with fluorochrome-conjugated antibodies against CD45, Tie2, AlphaV integrin, CD105, Thy1.1, Thy 1.2, 6C3 and CD200 for fractionation by fluorescence activated-cell sorting.
Extracted molecule
total RNA
Extraction protocol
RNA was isolated with RNeasy Micro Kit (Qiagen, Germantown, MD) as per manufacturer’s instructions. RNA was twice amplified with a RiboAmp RNA amplification kit (Arcturus Engineering, Mountain View, CA).
Label
biotin
Label protocol
Amplified cRNA was streptavidin-labeled, fragmented, and hybridized to Affymetrix 430-2.0 arrays as recommended by the manufacturer (Affymetrix, Santa Clara). Arrays were scanned with a Gene Chip Scanner 3000 (Affymetrix) running GCOS 1.1.1. software.
Hybridization protocol
standard Affymetrix protocol
Scan protocol
Arrays were scanned with a Gene Chip Scanner 3000 (Affymetrix) running GCOS 1.1.1. software.
Description
fore and hind limbs pooled litter
Data processing
Normalized Intenstity values were computed based on Gene Expression Commons Algorithum [ https://gexc.stanford.edu/]
