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Sample GSM7938598 Query DataSets for GSM7938598
Status Public on Dec 05, 2023
Title TP0001651E03_6_10.000000uM_DMEM_12h_TC00283174_E19
Sample type SRA
 
Source name MCF7 Cells
Organism Homo sapiens
Characteristics cell line: MCF7
media: DMEM + 10% HI-FBS
treatment: 12h exposure of 10 uM of Prochloraz
chemical name: Prochloraz
chemical sample id: TP0001651E03
chemical concentration: 10 uM
dose level: 6
exposure time: 12h
assay plate: TC00283174
assay plate well: E19
Treatment protocol DMSO-solubilized chemical stock solutions were received frozen from the US EPA ToxCast chemical inventory management contractor (EvoTec, Princeton, NJ) and stored at -80°C prior to dose plate preparation. An eight-point dilution series (1/2 log10 spacing) of test chemicals was prepared in a LabCyte Echo-qualified 384-well polypropylene (384PP) plate at 200x the desired nominal test concentration for screening (0.03 – 100 µM). Singleton wells of dosing solutions for each test chemical concentration were arrayed in columns 2 through 23 of the dose plate (Figure 1A). Transcriptomics reference chemicals (genistein, trichostatin A, sirolimus) were solubilized as 200x dosing solutions corresponding to nominal test concentrations of 10, 1 and 0.1 µM, respectively. Triplicate wells of dosing solutions for each of the transcriptomic reference chemicals were added to the dose plate (column 24) along with quadruplicate wells of pure DMSO as a vehicle control. At 24 h post-plating, 200 nL of 200x dosing solutions were transferred to the assay plates using a LabCyte Echo 550 acoustic dispenser. The final concentration of DMSO in all treatment, reference treatment and vehicle control wells was 0.5%. Well coordinates on each assay plate were uniquely randomized with respect to treatment so that any potential edge-well effects were distributed in an unbiased manner across all possible treatment conditions.
Growth protocol Five vials of cryopreserved MCF7 (HTB-22TM) cells were purchased from American Tissue Culture Collection, designated as passage 0 (P0) and stored in vapor phase liquid nitrogen prior to initial thawing and expansion. A passage 3 (P3) MCF7 cryostock was generated by thawing and pooling all five original source vials and culturing through three consecutive passages in complete growth medium (DMEM + 10% FBS + 1% PSG). At each passage, cells from different culture vessels were pooled prior to re-seeding for subsequent expansion. At P3, cells were cryopreserved at 4 million cells per mL in complete growth medium + 5% DMSO according to manufacturer’s protocol. MCF7 cultures used in all phases of this study were maintained in humidified incubators with 5% CO2 atmosphere and internal temperature of 37°C. For chemical screening, MCF7 cells were thawed and subjected to a uniform expansion protocol from P3 to P6 through increasingly larger sizes of tissue culture vessels (T25 to T75 to T225) prior to plating in 384-well format. The three cultures were initiated on consecutive days and subject to uniform handling procedures to ensure that dosing and sample collection also occurred on consecutive days. All chemical screening experiments were performed on P7 cells.
Extracted molecule total RNA
Extraction protocol Six hours after chemical treatment, HTTr assay plates were removed from the incubator and media in each assay well was drained to a residual volume of 10 µL using MultiFlo FX microdispenser equipped with a vacuum-driven aspiration manifold. To create cell lysates, 10 µL volumes of 2X BioSpyder lysis buffer were dispensed into each assay well using the same MultiFlo FX instrument equipped with 1 µL peristaltic pump dispensing cassette. Then, 20 µL volumes of UHRR, HBRR, BLDSMO, BLTSA and Lysis Buffer reference material were manually dispensed into duplicate wells of column 1 of each assay plate. The plates were sealed with an adhesive aluminum plate cover and incubated at room temperature, protected from light, for 30 minutes. The plates were then stored at -80°C prior to shipment to BioSpyder, Inc.
Samples were analyzed by BioSpyder using a custom-attenuated version of the TempO-Seq human whole transcriptome version 1 (hWTv1) assay, which includes 21,111 probes covering 19,287 genes. Lysates were processed as described in (House et al. 2017). In brief, 2 µL of each lysate was hybridized with 2 µL of detector oligos from the hWTv1 assay using the following thermal cycler protocol: 10 min at 70°C, followed by gradual decrease to 45°C over 49 min, terminating with 45°C incubation for 16-24 hours. Excess oligos were then removed via nuclease digestion (90 min at 37°C) and hybridized detector oligos were ligated (60 min at 37°) following respective additions of 24 µL TempO-Seq nuclease and ligation mixes. RNA/DNA duplexes were then heat-denatured and 10 µL of each ligation product was transferred to an amplification microplate containing 10 µL of PCR master mix per well. Ligation products were then uniquely labeled during product amplification (10 min at 37°C, 2 min at 95°C, 6 cycles of 95°C for 30 s, 54°C for 30 s, 72°C for 120 s, 16 cycles of 95°C for 30s and 72°C for 120s, followed by 72°C for 60 s) with well coordinate-specific “barcoded” primer pairs containing universal adaptors for sequencing. Samples were then pooled into a series of three sequencing libraries (1 for each plate). Pooled sequencing libraries were then distributed across multiple lanes of a HiSeq dual flow cell and analyzed on a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA).
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
 
Description TP0001651E03_6_10.000000uM_DMEM_12h
TC00283174_E19
Data processing Each FASTQ file was aligned to the probe sequences in the hWTv1 assay using HISAT2 v2.1.0 with spliced alignment disabled.
Aligned reads in SAM format were processed with SAMtools v1.9 to compute the number of uniquely aligned reads for each probe. Probe counts and associated meta-data for each well were stored for analysis using MongoDB v3.6.14.
Assembly: hWTv1
Supplementary files format and content: Processed data provided as a single table of counts for all probes x all samples
 
Submission date Dec 05, 2023
Last update date Dec 06, 2023
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
 
Platform ID GPL16791
Series (1)
GSE249377 Exploring the Effects of Experimental Parameters and Data Modeling Approaches on In Vitro Transcriptomic Point-of-Departure Estimates
Relations
BioSample SAMN38682853
SRA SRX22783797

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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