Fs 450 fluidics station

Genomic DNA was fragmented by incubating at 37°C for 1 min. in a 20 μl reaction containing 1 × One-Phor-All Plus Buffer (GE Healthcare) and 0.01 units DNase I (GE Healthcare) as previously described (Jackson et al., 2011 (link)), and further modified by Tall et al. (2015 (link)). The fragmented DNA was heat-inactivated at 99°C for 15 min, and was 3′-end labeled by adding 4 μl of 5 × terminal transferase buffer (Promega), 1 μl 1 mM biotin-11-ddATP (PerkinElmer NEL508), and 2 μl (60 units) of terminal transferase enzyme (Promega). The labeling was carried out for 4 h at 37°C followed by heat inactivation at 98°C for 1 min.
Hybridizations were performed according to the Affymetrix GeneChip Expression Analysis Technical Manual for a 49-format array (Affymetrix, 2014 ). Following hybridization, wash and stain procedures were carried out on an Affymetrix FS-450 fluidics station using the mini_prok2v1_450 fluidics script (Affymetrix, 2014 ). Reagents for washing and staining were prepared according to the GeneChip® Expression Analysis Technical Manual (Affymetrix, 2014 ). The following modifications were made to the wash and stain procedure: Streptavidin solution mix (vial 1) was replaced with SAPE solution mix (Life Technologies, Grand Island, NY). Arrays were scanned using Affymetrix GeneChip® Scanner 3000 running on AGCC software.

100 ng of total RNA from PBMC from 4 controls and 5 PE at week 22–24 was subjected to GeneChip HT One-Cycle cDNA Synthesis Kit and GeneChip HT IVT Labeling Kit, following the manufacturer’s protocol for whole genome gene expression analysis (Affymetrix, Santa Clara, CA). Labeled and fragmented single stranded cDNAs were hybridized to the GeneChip Human Gene 1.0 ST Arrays (28,869 transcripts) (Affymetrix). The arrays were washed and stained using FS-450 fluidics station (Affymetrix). Signal intensities were detected by Hewlett Packard Gene Array Scanner 3000 7G (Hewlett Packard, Palo Alto, CA). The scanned images were processed using Affymetrix GeneChip Command Console (AGCC). The CEL files were imported into Partek Genomics Suite software (Partek, Inc. MO). Robust microarray analysis (RMA) was applied for normalization. Differentially expressed genes between groups were identified using one-way ANOVA analysis. Cluster analysis were generated in Partek Genomics Suite. Further bioinformatics analysis was conducted on the significant genes to identify functional significance by means of Ingenuity Pathways Analysis (Ingenuity Systems, Redwood City, CA).