Gene expression arrays

Total RNA was isolated from cells grown under different assay conditions. The quality and quantity of RNA was assessed spectrophotometrically by a Nanodrop and Bioanalyzer. Equal amounts of RNA samples were used to prepare cDNA library and processed for transcriptomic analysis using Illumina Gene Expression arrays. The raw data obtained from hybridization to illumina HT-12 array, was quantile normalized followed by baseline transformation to median of all the samples using GeneSpring GX 12.5 software. The differentially expressed genes were identified across the samples using volcano plot with a fold change threshold of 1.5 and t test p value threshold adjusted for false discovery rate less than 0.001 for statistical significance. Hierarchical clustering of differentially expressed genes in treated vs. control conditions was done using Euclidian algorithm with Centroid linkage rule to identify gene clusters whose expression levels were significantly reproduced across the replicates. Differentially expressed genes were subjected to biological significance analysis by GOElite tool to determine enriched biological pathways. Over representation Analysis (ORA) of significant biological categories (GeneOntology and Pathway) involving differentially expressed transcripts was performed and a network was modeled. GEO accession number for the microarray data: GSE125317.

We used eQTL summary data from the Consortium for the Architecture of Gene Expression (CAGE, n = 2,765 in peripheral blood) and the GTEx Consortium v7 release (n = 369 from whole blood). Briefly, Lloyd-Jones et al. (2017) (link) performed this analysis of 2,765 samples from peripheral blood, with gene expression data observed from Illumina gene expression arrays (38,624 gene expression probes) and SNP genotype data imputed to the 1000 Genomes Phase 1 Version 3 reference panel (∼8 million SNPs). Information about tissue collection, genotyping, RNA quantification, and statistical analysis can be found in the original study of Lloyd et al. (2017). The GTEx project (GTEx Consortium, 2013 (link)) contained samples from 44 healthy tissues of 20- to 70-year-old human postmortem donors. For GTEx eQTL data (v7), whole blood tissues of 369 individuals were used, and gene expression levels measured by RNA-seq. SNP genotyping was performed using the Illumina OMNI SNP Arrays.