biostudies-arrayexpressDr. Julian MarschnerHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15058The ligand dependent nuclear receptor subtypes RXRa, RXRb, and RXRc are key mediators in mammalian homestasis and in response to pathological conditions, e.g. inflammation and neurodegeneration. We allocated a set of RXR pan-agonists including the established agonist Bexarotene, a drug used as second line treatment for cutaneous T-cell lymphoma, and four additional chemical tools (JP147, JP175, PH299, S169), that surpass Bexarotene in terms of physicochemical properties and - more importantly - selectivity within the family of nuclear receptors. The ligand dependent nuclear receptor subtypes RXRa, RXRb, and RXRc are key mediators in mammalian homestasis and in response to pathological conditions, e.g. inflammation and neurodegeneration. We allocated a set of RXR pan-agonists including the established agonist Bexarotene, a drug used as second line treatment for cutaneous T-cell lymphoma, and four additional chemical tools (JP147, JP175, PH299, S169), that surpass Bexarotene in terms of physicochemical properties and - more importantly - selectivity within the family of nuclear receptors. We investigated changes in RNA expression profiles in HEK293T cells upon stimulation with said modulatirs. HEK293T cells were seeded at a density of 3 x 10^6 cells per well in 12-well plates and complete culture medium. After 24 h, medium was changed to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS for synchronization over 24 h. Medium was changed again to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS, additionally containing either test compounds (1 µM) in 0.1% DMSO or 0.1% DMSO. After 17 h, cells were harvested and total RNA was isolated using E.Z.N.A. Total RNA Kit I (Omega Bio-tek) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop One UV–vis spectrophotometer (Thermo Fisher Scientific) at 260/280 nm. We isolated total RNA from ~ 300.000 cells per sample using silica gel columns and submitted the samples to RNA sequencing at Novogene (United Kingdom).biostudies-arrayexpressNucleic Acid Extraction - Total RNA was isolated using E.Z.N.A. Total RNA Kit I (Omega Bio-tek) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop One UV–vis spectrophotometer (Thermo Fisher Scientific) at 260/280 nm. Novoegene repeated the isolation using poly-T oligo-attached magnetic beads.Sequencing - Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount. The clustering of the index-coded samples was performed according to the manufacturer’s instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.Sample Treatment - After 24 h, medium was changed to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS for synchronization over 24 h. Medium was changed again to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS, additionally containing either test compounds (1 µM) in 0.1% DMSO or 0.1% DMSO. After 17 h, cells were harvested the cells.Sample Collection - Samples were collected by aspirating stimulation medium and washing each well with PBS once. Thereafter cells were lysed using the lysis buffer from E.Z.N.A. Total RNA Kit I (Omega Bio-tek) according to the manufacturer’s instructions.Library Construction - After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.Growth Protocol - HEK293T cells were seeded at a density of 3 · 106 cells per well in 12-well plates and complete culture medium.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The biological replicates were normalized using theDESeq2 package in R, a negative binominal distribution was assumed to calculate p-values, and the Benjamini-Hochberg procedure was apllied to control the false discovery rate.UnknownTranscriptomicsGenomicsProteomicsE.Z.N.A. Total RNA Kit I (Omega Bio-tek)NAIllumina NovaSeq 6000unknownRNA-seq of coding RNAHomo sapiensDr. Julian MarschnerfalseInvestigating RXR dependent changes in mRNA expression by using novel chemical modulatorsThe ligand dependent nuclear receptor subtypes RXRa, RXRb, and RXRc are key mediators in mammalian homestasis and in response to pathological conditions, e.g. inflammation and neurodegeneration. We allocated a set of RXR pan-agonists including the established agonist Bexarotene, a drug used as second line treatment for cutaneous T-cell lymphoma, and four additional chemical tools (JP147, JP175, PH299, S169), that surpass Bexarotene in terms of physicochemical properties and - more importantly - selectivity within the family of nuclear receptors. The ligand dependent nuclear receptor subtypes RXRa, RXRb, and RXRc are key mediators in mammalian homestasis and in response to pathological conditions, e.g. inflammation and neurodegeneration. We allocated a set of RXR pan-agonists including the established agonist Bexarotene, a drug used as second line treatment for cutaneous T-cell lymphoma, and four additional chemical tools (JP147, JP175, PH299, S169), that surpass Bexarotene in terms of physicochemical properties and - more importantly - selectivity within the family of nuclear receptors. We investigated changes in RNA expression profiles in HEK293T cells upon stimulation with said modulatirs. HEK293T cells were seeded at a density of 3 x 10^6 cells per well in 12-well plates and complete culture medium. After 24 h, medium was changed to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS for synchronization over 24 h. Medium was changed again to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS, additionally containing either test compounds (1 µM) in 0.1% DMSO or 0.1% DMSO. After 17 h, cells were harvested and total RNA was isolated using E.Z.N.A. Total RNA Kit I (Omega Bio-tek) according to the manufacturer’s instructions. RNA concentration and purity were assessed using a NanoDrop One UV–vis spectrophotometer (Thermo Fisher Scientific) at 260/280 nm. We isolated total RNA from ~ 300.000 cells per sample using silica gel columns and submitted the samples to RNA sequencing at Novogene (United Kingdom).2025-05-01T00:00:00Z2025-04-17T15:33:33.256Z2025-04-17T15:33:33.256ZE-MTAB-15058ERP171780EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_0003969