Labelled using protocol described in Affymetrix manual "3' IVT Express Kit User Manual"
Hybridization protocol
Biotin Labelled cRNA using "Affymetrix GeneChip Hybridization, Wash and Stain Kit", according to guidelines set out by Affymetrix, Santa Clara, CA
Scan protocol
Scanned according to guidelines set out by Affymetrix, Santa Clara, CA.
Description
Seeds were dried after harvesting and cold-treated for minimum 7 days at 4°C 6 minutes shaking in 5% hypochloric acid, 0.05% Tween-80 solution. Subsequent washes with ddH2O (3x) and over night drying. Cold treated seeds were surface-sterilized in 5% hypochloric acid and 0,05% Tween-80 for 6 minutes, washed and air-dried overnight. 50 seeds were sawn in a regular matrix on petri dishes containing semisolid germination medium with 0.8% agar and grown for 17 days in Percival growth chambers at 16h light/8h dark cycles and 21°C. 8 50/plate 21 °C average, 21 °C day, 21 °C night ~100% % average, ~100% % day, ~100% % night Own medium: MS macro 50ml; B5 micro 1ml; ammonium iron citrate 50mg; sucrose 10g; MES 350mg; 8% Agar 450ml
Data processing
Raw data from the microarrays was normalized at probe-level using MAS5 algorithm. The detection calls (present, marginal, absent) for each probe set was obtained using the GCOS system
