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Series GSE200845 Query DataSets for GSE200845
Status Public on Apr 18, 2022
Title Combining phenotypic profiling and targeted RNA-Seq reveals linkages between transcriptional perturbations and chemical effects on cell morphology: Retinoic acid as an example
Organism Homo sapiens
Experiment type Expression profiling by high throughput sequencing
Other
Summary The United States Environmental Protection Agency has proposed a tiered testing strategy for chemical hazard evaluation based on new approach methods (NAMs). The first tier includes in vitro profiling assays applicable to many (human) cell types, such as high-throughput transcriptomics (HTTr) and high-throughput phenotypic profiling (HTPP). The goals of this study were to: (1) harmonize the seeding density of U-2 OS human osteosarcoma cells for use in both assays; (2) compare HTTr- versus HTPP-derived potency estimates for 11 mechanistically diverse chemicals; (3) identify candidate reference chemicals for monitoring assay performance in future screens; and (4) characterize the transcriptional and phenotypic changes in detail for all-trans retinoic acid (ATRA) as a model compound known for its adverse effects on osteoblast differentiation. The results of this evaluation showed that (1) HTPP conducted at low (400 cells/well) and high (3000 cells/well) seeding densities yielded comparable potency estimates and similar phenotypic profiles for the tested chemicals; (2) HTPP and HTTr resulted in comparable potency estimates for changes in cellular morphology and gene expression, respectively; (3) three test chemicals (etoposide, ATRA, dexamethasone) produced concentration-dependent effects on cellular morphology and gene expression that were consistent with known modes-of-action, demonstrating their suitability for use as reference chemicals for monitoring assay performance; and (4) ATRA produced phenotypic changes that were highly similar to other retinoic acid receptor activators (AM580, arotinoid acid) and some retinoid X receptor activators (bexarotene, methoprene acid). This phenotype was observed concurrently with autoregulation of the RARB gene. Both effects were prevented by pre-treating U-2 OS cells with pharmacological antagonists of their respective receptors. Thus, the observed phenotype could be considered characteristic of retinoic acid pathway activation in U-2 OS cells. These findings lay the groundwork for combinatorial screening of chemicals using HTTr and HTPP to generate complementary information for the first tier of a NAM-based chemical hazard evaluation strategy.
 
Overall design We evaluated the Templated Oligo with Sequencing Readout (TempO-Seq) assay for High-throughput transcriptomic screening of a small set of environmental chemicals. This assay yields sequencing reads of exactly 50 base pairs that can be rapidly aligned to generate gene counts, and is compatible with cell lysates prepared in multiwell format. The version of the TempO-Seq assay we evaluated provides nearly whole transcriptome coverage (>20,000 genes). This study encompasses 2 replicates of a 384-well plate design. The majority of wells contain U-2 OS cells exposed to 11 test chemicals at 7 different concentrations (two replicate per test chemical, concentration, and plate), and additional reference samples and controls. Controls include DMSO vehicle treatments (22 per plate). Reference samples include bulk lysate MCF7 samples (2 DMSO treated and 2 TSA treated samples per plate), reference chemical treatments (3 chemicals at single conc each, per plate), and vendor-provided reference RNA mixtures (UHRR and HBRR).
 
Contributor(s) Nyffeler J, Willis C, Harris FR, Taylor LW, Judson R, Everett LJ, Harrill JA
Citation(s) 35483669
Submission date Apr 14, 2022
Last update date Jul 18, 2022
Contact name Logan J Everett
Organization name U.S. Environmental Protection Agency
Department Office of Research and Development
Lab Center for Computational Toxicology and Exposure
Street address 109 T.W. Alexander Dr
City RTP
State/province North Carolina
ZIP/Postal code 27711
Country USA
 
Platforms (1)
GPL16791 Illumina HiSeq 2500 (Homo sapiens)
Samples (372)
GSM6044924 UHRR_TC00000651_A01
GSM6044925 UHRR_TC00000651_A02
GSM6044926 U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A10_7_0.100000uM_TC00000651_A03
Relations
BioProject PRJNA826904

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Supplementary file Size Download File type/resource
GSE200845_hWTv2_u2os_probes.csv.gz 1.2 Mb (ftp)(http) CSV
GSE200845_httr_u2os_pilot_GEO_count_data.csv.gz 9.0 Mb (ftp)(http) CSV
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Processed data are available on Series record
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