genotype: Moroc9-75 trait for drought tolerance: drought tolerance (sensitive) tissue: flag leaves treatment: 3 d after drought stress
Seven flag leaves of a replication for each genotype were harvested at 3 d after reach 10% of AWC in the soil to constitute a single biological replicate. These flag leaves were employed for RNA isolation by using Trizol reagent following the manufacturer’s protocol (Invitrogen, Karlsruhe, Germany). The RNA was further purified using RNeasy Kit (Qiagen, Hilden, Germany). RNA yield and quality were determined by using an Agilent 2100 Bioanalyzer (Agilent Techologies, Boblingen, Germany).
5 µg of high quality total RNA were reverse transcribed using the One-Cycle cDNA Synthesis Kit (Affymetrix, Santa Clara, CA, USA). The resulting double-stranded cDNA was used as a template to generate biotin-tagged cRNA from an in vitro transcription reaction (IVT), using Affymetrix IVT Labeling Kit as Expression Analysis Technical Manual (2001, Affymetrix)
Biotin-tagged cRNA was fragmented to strands of 35 to 200 bases in length, and 15 µg of fragmented cRNA was used for each hybridization. Barley 1 GeneChips were hybridized for 16 h at 45°C with rotation in a GeneChip® Hybridization Oven 640 (Affymetrix, Santa Clara, CA, USA). The arrays were washed and stained with R-phycoerythrin streptavidin in a Fluidics Station.
The Arrays were subsequently scanned with Affymetrix GeneChip® Scanner 3000
All scanned data from barley 1 GeneChips were processed by robust multiarray average (RMA) (Irizarry et al., 2003) using ArrayAssist software version 3.4 (Stratagene, La Jolla, CA, UAS)
Normalized expression values were computed from raw CEL files by first applying the RMA model of probe-specific correction of perfect match probes. The algorithm consists of three steps—a model-based background correction stage neutralizes the effects of background noise and the processing artifacts, a subsequent quantile normalization stage aligns expression values to a common scale, and finally, an iterative median polishing procedure summarizes the data and generates a single expression value for each probe set. Resulting RMA expression values were log2-transformed. Average log signal intensity values of three biological replicates for each sample were then computed and these were used for further analysis. To identify differentially expressed genes, student's t-test was applied with a maximal FDR (false discovery rate) of 5% (Benjamini-Hochberg algorithm). RMA data for each individual Affymetrix chip not available. Thus the Sample data tables instead report MAS5.0-processed data. A table of the average, log2 RMA signal intensity values of three biological replicates for each sample is linked as a supplementary file on the GSE15970 record.