Feature extraction software version 9

For microarray hybridization, rice RNA samples were subjected to quality control using an Agilent Bioanalyzer 2100. Only those showing no degradation and clear 28S ribosomal RNA (rRNA) and 18S rRNA peaks were used. A two-color microarray-based analysis with Low Input Quick Amp Labeling kit and 4×44 k 60-mer microarrays (Agilent; Böblingen, Germany) was carried out according to the company’s instructions. The data extractions were performed with Feature Extraction Software version 9.5 (Agilent; Böblingen, Germany) and GeneSpring software (Agilent; Böblingen, Germany). For each experiment three biological replicates were performed, and for hybridization one dye-swap and a technical replicate were included. Genes showing equal or larger than 1.5-fold up- or down-regulation in all three experiments were regarded as differently regulated. Data were deposited at GEO (Gene Expression Omnibus) (GSE136706 and GSE136707).

The fluorescence intensities were extracted using the Agilent Feature Extraction Software Version 9.5, using the protocol GE2-v5_95_Feb07 without spike-in controls. The probes were filtered using the IsFound, IsFeatNonUnif, IsBGNonUnif, ISFeatPopnOL, and IsBGPopnOL flags, and discarded if the first flag had a value of 0, or any of the others had a value of 1. We used the ‘LogRatio’ value for each probe, and all probes which were on the sense strand of the coding region of a gene were assigned to the gene. The values were averaged across all probes for a gene, and across the two biological replicates for each experiment.
We ran iPAGE [13 (link)] in continuous mode with various numbers of bins, which did not significantly change the categories identified. The ‘GO annotation’ module was used for the data shown in Fig 2, and the ‘Transcription factor regulon’ module was used for the data shown in Fig 6.

Global gene expression was determined during log-phase growth of B. breve UCC2003 in mMRS supplemented with either LNT, LNnT, LNB, lactosamine-HCl or lactose. The obtained transcriptome was compared to that determined for log-phase B. breve UCC2003 cells when grown in mMRS supplemented with ribose. DNA microarrays containing oligonucleotide primers representing each of the 1864 identified open reading frames on the genome of B. breve UCC2003 were designed and obtained from Agilent Technologies (Palo Alto, Ca., USA). Methods for cell disruption, RNA isolation, RNA quality control, complementary DNA synthesis and labelling were performed as described previously68 (link). Labelled cDNA was hybridized using the Agilent Gene Expression hybridization kit (part number 5188-5242) as described in the Agilent Two-Colour Microarray-Based Gene Expression Analysis v4.0 manual (publication number G4140-90050). Following hybridization, microarrays were washed in accordance with Agilent’s standard procedures and scanned using an Agilent DNA microarray scanner (model G2565A). Generated scans were converted to data files with Agilent’s Feature Extraction software (Version 9.5). DNA-microarray data were processed as previously described69 (link)70 (link)71 (link). Differential expression tests were performed with the Cyber-T implementation of a variant of the t-test72 (link).