Samples were frozen in liquid nitrogen and stored at -80°C until use.
Fifty to 100 g seeds of each variety were micro-malted at Busch Agricultural Resources, LLC, Fort Collins, CO. The micromalting conditions were as follows: steeping at 12°C for 37 h, germination at 12°C for 5 h, 17°C for 65 h, 18°C for 23 h, and kilning stages consisting of 55°C for 10 h, 60°C for 4 h, 68°C for 3 h, 80°C for 2 h and 90°C for 3 h.
Frozen barley malt was ground and incubated with RNA purification reagent from Invitrogen (Carlsbad, CA) at room temperature for 10 minutes. Total RNA was extracted using Qiagen RNeasy Plant Mini Kit with RNase Free/DNase (Qiagen, Valencia, CA) following the manufacturer's instructions.
The BioArray High-Yield RNA Transcript labeling kit (Enzo Diagnostics) was used to synthesize biotin-labeled cRNA from template cDNA by in vitro transcription.
10 µg labeled, fragmented cRNA was hybridized at 45°C with rotation for 16 h in an Affymetrix GeneChio Hybridization Oven 320 on Affymetrix Barley1 GeneChip arrays. The arrays were washed and stained using streptavidin phycoerythrin on an Affymetrix Fluidics Station 400.
GeneChips were scanned using the GeneChip Scanner 3000.
Probe synthesis, labeling and hybridization were perfomed according to manufacturer's protocols (Affymetrix, Santa Clara, CA) at the UCI Microarray Core Facility, Irvine, CA.
Different quality control checks were performed including inspection of hybridized images, boxplots and histograms of log2(PM) values, examination of hybridization and PolyA controls. Based on the results of the quality control screening, some chips were discarded from the analysis. Twenty-two arrays were retained for further analysis, with two replications per cultivar per time point, with the exception of Harrington day 1, where one of the replicates was discarded. Data analysis was carried out using Bioconductor in R (Gentleman et al. 2004). Data preprocessing and summarization were performed using Robust Multichip Average (RMA) (Irizarry et al. 2003).