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
After sterilization, seeds were incubated in sterile water in the dark for 5 days at 4°C prior to cultivation Seed were washed briefly (4 min) in 70% ethanol followed by an 8 min treatment with bleach. Afterwards seeds were washed 3 times with sterile deionised water. After final wash, seeds were resuspended in sterile deionised water and subjected to stratification Sterilized and stratified seeds were grown in 300 ml Erlenmeyer flasks containing 100 ml of sterile liquid ATS medium. Plants were cultivated on a shaker for 7 days at 90 rpm and 22 °C under constant white fluorescent light. For the last 24 hours of cultivation the light was filtered by yellow plexiglas (yellow 2208)to prevent photodegradation of IAA. Reference for ATS medium: Lincoln C, Britton JH, Estelle M (1990) Growth and Development of the axr1 Mutants of Arabidopsis. Plant Cell 2: 1071-1080 autoclaved 22 °C average, 22 °C day, 22 °C night 70 % average, 70 % day, 70 % night Own medium: ATS medium according to Lincoln et al. (1990) Constant light
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
