Glucocorticoid gene expression data

We analyzed RNA-sequencing data from a set of experiments developed to study glucocorticoid receptors (GRs) in the human adenocarcinoma and lung model cell line, A549 [6]. There was an original exposure data set of 4 replicates in which cells were stimulated by the glucocorticoid dexamethasone (dex), and gene expression was profiled at {0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12} hours of dex stimulation. There was also an unperturbed data set of 3 replicates in which cells were exposed to dex for 12 hours, after which the conditioned media was replaced and dex removed. Gene expression was profiled at {0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12} hours after dex removal. We integrated the original exposure and unperturbed data into a joint data set with 7 replicates. The original exposure data set is available at the Gene Expression Omnibus (GEO), with reference numbers listed for the rows that list “RNASeq” as the Assay under the column “Experiment_GEO_Series” in Supplementary Table 3 of [6]. The GEO accession numbers for time points {0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12} of the original exposure data set are {"type":"entrez-geo","attrs":{"text":"GSE91305","term_id":"91305"}}GSE91305, {"type":"entrez-geo","attrs":{"text":"GSE91198","term_id":"91198"}}GSE91198, {"type":"entrez-geo","attrs":{"text":"GSE91311","term_id":"91311"}}GSE91311, {"type":"entrez-geo","attrs":{"text":"GSE91358","term_id":"91358"}}GSE91358, {"type":"entrez-geo","attrs":{"text":"GSE91303","term_id":"91303"}}GSE91303, {"type":"entrez-geo","attrs":{"text":"GSE91243","term_id":"91243"}}GSE91243, {"type":"entrez-geo","attrs":{"text":"GSE91281","term_id":"91281"}}GSE91281, {"type":"entrez-geo","attrs":{"text":"GSE91229","term_id":"91229"}}GSE91229, {"type":"entrez-geo","attrs":{"text":"GSE91255","term_id":"91255"}}GSE91255, {"type":"entrez-geo","attrs":{"text":"GSE91284","term_id":"91284"}}GSE91284, {"type":"entrez-geo","attrs":{"text":"GSE91222","term_id":"91222"}}GSE91222, and {"type":"entrez-geo","attrs":{"text":"GSE91212","term_id":"91212"}}GSE91212, respectively. The unperturbed data set is available at GEO accession number GSE144662.

We selected 2768 genes for analysis, which had average expression > 2 Transcripts Per kilobase Million (TPM) and were differentially expressed in the original exposure data. A gene was called differentially expressed if its expression at any time point differed from its expression at time 0, ascertained by running edgeR (FDR ≤ 0.05) [6]. We added NR3C1, which encodes the glucocorticoid receptor (GR). NR3C1 was not found to be differentially expressed at FDR ≤ 0.05.

After genes were selected, gene expression TPM were log-normalized and corrected for surrogate variables using SVAseq [85]. Each gene’s temporal profile was centered to have mean zero across time. In the original exposure data, all replicates besides replicate 1 had a measurement for each time point. Replicate 1 was missing time points 5 and 6 hrs, so we imputed these values using a linear interpolation from time points 4 and 7 hrs in the log-transformed, surrogate-corrected space.