GSE172478
Transcriptomic analysis of corticosteroids-treated hiPSC-derived RPE cells
The aim of this study was to analyze the pan-transcriptional regulations induced in hiPSC-derived RPE (iRPE) cells by corticoids with specific inhibitors of their receptors to decipher specific genes regulated by MR or GR activation. Cells were seeded at P3 in cell culture plastic dishes. On day 35, one week prior to corticosteroids treatments, RDMw/oA medium was remove and iRPE cells were incubated in experimental corticosteroid-free medium (DMEM, high glucose, HEPES, no phenol red, 10% Fetal Bovine Serum, charcoal stripped). On day 42, iRPE cells were treated for 24 h with the following corticosteroids treatments: aldosterone (10-7 M), cortisol (10-7 M) and cortisol (10-7 M) plus RU-486 (10-5 M).  As corticosteroids were dissolved in ethanol (EtOH) or methanol (MeOH) control cells were treated with 0.1% EtOH or MeOH  in medium. Total RNA samples extracted from iRPE were sequenced at the iGenSeq transcriptomic platform of the Brain and Spine Institute (ICM, Paris, France). RNA quality was checked by capillary electrophoresis (Agilent 2100 Bioanalyzer system) and RNA with integrity numbers (RIN) ranging from 7.8 to 8.2 was accepted for library generation. Quality of raw data has been evaluated with FastQC. Librairies were prepared with Roche KAPA mRNA HyperPrep kit and sequenced with the Illumina NextSeq 500 Sequencing system using NextSeq 500 High Output Kit v2 (150 cycles), 400 millions of reads, 50Gbases. Star v2.5.3a has been used to align reads on reference genome hg19 using standard options. Quantification of gene and isoform abundances has been done with rsem   1.2.28, prior to normalization on library size with edgeR bioconductor package. Finally, differential analysis has been also conducted with edgeR. Multiple hypothesis adjusted p-values were calculated with the Benjamini-Hochberg procedure to control FDR. We identified genes differentially regulated by MR and GR, and those regulated by both MR and GR activation in hiPSC-RPE cells. Overall design: mRNA profiles of corticosteroids-treated hiPSC-RPE cells