Barley Transcriptome Analysis Protocol

For transcriptome analysis, an Agilent 8×60K customized barley array was used (Kohl et al., 2015 (link)). The design is available at EMBL-EBI ArrayExpress, accession E-MTAB-3040. Total RNA was extracted from freshly isolated pericarps with a Spectrum™ Plant Total RNA Kit (Sigma Aldrich, Steinheim, Germany) and integrity was confirmed using Bioanalyser (Agilent Technologies). A 100ng aliquot of RNA was used for cRNA synthesis and Cy3 labelling via a Low Input Quick Amp Labeling Kit (Agilent Technologies). Labelling efficiency, amount, and quality of cRNA were assured using an ND-1000 Spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and Bioanalyser. A 600ng aliquot of labelled cRNA was used for fragmentation and array loading (Gene Expression Hybridisation Kit, Agilent Technologies). Hybridization was carried out for 17h at 65 °C. Arrays were scanned at 5 μm resolution (Agilent Technologies Scanner G2505C) and images were evaluated (determination of spot intensities, background correction) with Feature Extraction V11.5 (Agilent Technologies).