Whole human genome microarray kit

In order to determine gene expression profiles, 44 K DNA microarrays (Whole Human Genome Microarray Kit, Agilent Technologies, cat no. G4112F, Santa Clara, CA, USA) were used. The procedures for hybridization using the fluorescent dye Cy3 followed the manufacturer’s protocols (One-Color Microarray-Based Gene Expression Analysis—Quick Amp Labeling). The microarray images were captured by the reader Agilent Bundle, according to the parameters recommended for bio-arrays and extracted by Agilent Feature Extraction software version 9.5.3. Spots with two or more flags (low intensity, saturation, controls, etc.) were considered as NA, that is, without valid expression value. The R software version 2.11.1 and an in-house script were used for: i) excluding transcript spots presenting one or more NAs; iii) converting gene expression values to log base 2. Through this procedure the gene expression matrix with only expressed transcripts were obtained. Data normalization was performed using R software and the Lowess method [16 ]. TMEV software version 4.6.1 and t-test with p<0.05 and fold-change value ≥ 2.0 was used for obtaining the differentially expressed genes for the comparison (HRSV versus HRV). All microarray raw data have been deposited in Expression Omnibus (GEO) public database under accession number GSE124124.

Dual dye microarray using the Whole Human Genome Microarray Kit (Agilent Technologies, Inc., Santa Clara, CA) was carried out following the vendor’s protocol. We labeled 12.5 ng amplified RNA with biotin and the same amount of reference RNA with fluorescein. Samples were hybridized to Agilent 4 × 44 k slides and incubated for 16 h at 55°C. A protocol with a series of washes was carried out for tagging Cy-3 and Cy-5 dyes to reference and sample RNA, respectively. The slides were scanned using Agilent Technologies Scanner G2505C US09493743 and feature extracted using the software v. 10.7 (Agilent, Inc., CA). The assay quality was verified by assessing no alteration of a set of housekeeping genes across the experimental parameters discussed in the supplementary section.
We have submitted the microarray data to the Gene Expression Omnibus (GEO) and this can be searched for using the GEO accession number: GSE54213.

In order to determine gene expression profiles, 4 × 44 K DNA microarrays (Whole Human Genome Microarray Kit, Agilent Technologies, cat no. G4112F, Santa Clara, CA) were used. The procedures for hybridization using the fluorescent dye Cy3 followed the manufacturer’s protocols (One-Color Microarray-Based Gene Expression Analysis—Quick Amp Labeling). The images were captured by the reader Agilent Bundle according to the parameters recommended for bioarrays and extracted by Agilent Feature Extraction software version 9.5.3. (https://www.agilent.com/). Spots with two or more flags (low intensity, saturation, controls, etc.) were considered as NA, that is, without valid expression value. An in-house algorithm in R environment (version 3.4.4⁹³ ) was used for excluding transcript spots presenting one or more NAs and for converting gene expression values to log base 2. Through this procedure we identified 8,104 Gene Ontology (GO) annotated genes. Data normalization was performed using limma package^{94 ,95} in R environment (version 3.4.4⁹³ ). All microarray raw data have been deposited in GEO public database (http://www.ncbi.nlm.nih.gov/geo), a MIAME compliant database, under accession number GSE163296.