After dissociating the limbs into single cell suspensions, GFP-positive (ZPA) and GFP-negative (rest of the limb) cells were purified by FACS.
Growth protocol
Shhgfpcre heterozygotes were harvested at E10.5.
Extracted molecule
total RNA
Extraction protocol
Qiagen RNeasy according to manufacturer's instructions
Label
biotin
Label protocol
Biotinylated cRNA was prepared according to the two-cycle Affymetrix protocol from 50ng total RNA (Expression Analysis Technical Manual, 2003, Affymetrix).
Hybridization protocol
Following fragmentation, 15 ug of cRNA was hybridized for 16 hr at 45C on Mu430 2.0 A Array. GeneChips were processed on GeneChip fluidics station F450.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
Description
gene expression from ZPA cells
Data processing
The data were analyzed with GCOS 1.3 using Affymetrix default analysis settings and global scaling as normalization method. The trimmed mean target intensity of each array was arbitrarily set to 500.
