Agilent barley gene expression microarray

The synthesis, labeling, and hybridization of cDNA and cRNA were performed by the Genomics Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany. The microarray data were analyzed using GeneSpring GX 13.0 software (Agilent Technologies) as described earlier (Daszkowska-Golec et al., 2017 (link)). A gene was considered to be differentially expressed when the level of its expression differed between the analyzed conditions by at least two times (fold change (FC) ≥ 2; P ≤ 0.05 after FDR correction). The annotation of the Agilent Barley Gene Expression Microarray (Agilent Technologies) was performed against IBSC_v2 of barley genome deposited in Ensembl Plants (v. 45). Functional annotation of differentially regulated genes was carried out using Ensembl Plants tools and the IPK Barley BLAST Server as references (https://plants.ensembl.org/index.html, https://webblast.ipk-gatersleben.de/barley_ibsc/). Three biological replications were used for microarray expression analysis (each biological replicate represented leaf of one seedling).

The Agilent Barley Gene Expression Microarray (Agilent Technologies) was used for the global analysis of leaf and root transcriptomes of the WT cv. ‘Karat’ and the rhl1.a mutant. First, a de novo annotation of the array was done. Using a BLAST-based combinatorial strategy, 18 000 array probes were mapped to cDNAs that represented the 11 340 unique high-confidence (HC) genes that have been annotated in the barley genome. Taking into account that the estimated number of HC barley genes is 24 318 (International Barley Genome Sequencing Consortium, 2012 (link)), the Agilent Barley Gene Expression Microarray represents about 47% of barley genes. The main objective of the annotation of the Agilent Barley Gene Expression Microarray via the mapping of the array to the HC genes was to use their broad annotations provided by the PLAZA database (http://bioinformatics.psb.ugent.be/plaza/versions/plaza_v3_monocots; Proost et al., 2015 (link)) which include Gene Ontology (GO) annotation, protein domains, homologous gene families, and the prediction of orthologous genes. This annotation of barley genes provided by PLAZA makes it the most comprehensive source of information, and we subsequently used it for all of the bioinformatic annotation of barley genes, including barley/Arabidopsis cross-species analysis.