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| Status |
Public on Mar 06, 2018 |
| Title |
VEH1 |
| Sample type |
SRA |
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| Source name |
Trophoblast stem cells, Methanol (vehicle control)
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| Organism |
Macaca mulatta |
| Characteristics |
cell line: Trophoblast stem cells (TSCs) (line 119-T)
treatment: Methanol (vehicle control)
passages: 3
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| Treatment protocol |
The cells were grown to confluence and passaged weekly by rinsing wells with PBS, incubating with 1.0 mL trypsin-EDTA, and transferring 20% of cell suspension to each new well. Treatments began when cells first reached confluence and continued for 4 weeks, including during passage.
Chemicals for testing were made as 1000x stocks in methanol and were added to aliquots of TSC maintenance medium up to 48 hours before use and stored at 4°C until use. Chemicals were obtained from Sigma (DEHP: Bis(2-ethylhexyl) phthalate – 47994; PFOA: Perfluorooctanoic acid – 171468; ATR: Atrazine – 45330; TBT: Tributyltin chloride – 442869; BPA: Bisphenol A – 133027). In addition to untreated control, treatment groups included a vehicle control (methanol) and each of five EDCs at environmentally relevant doses [10 nM BPA (36 nM fetal serum), 5 uM DEHP (7.7uM pubertal serum), 30 uM ATR (up to 7 uM drinking water reported), 100 nM PFOA (average 94 nM >12 year old serum), and 25 nM TBT (0.17 – 534 adult nM serum).
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| Growth protocol |
Trophoblast stem cells (TSCs) (line 119-T) were isolated previously from rhesus monkey blastocysts and characterized using a panel of antibodies to detect TSC biomarkers. TSCs were maintained in DMEM/F- 12 (Invitrogen) supplemented with 15% fetal bovine serum (FBS; Hyclone), 1% minimum essential medium (MEM) nonessential amino acids (Invitrogen), 1 mM glutamine (Sigma), 0.1 mM β -mercaptoethanol, and 1X penicillin/streptomycin sulfate (Invitrogen) on plates coated with human placental collagen (Sigma). Medium was changed daily.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were detached with 0.25% trypsin-EDTA (Gibco). They were then centrifuged at 3000 x g for 3 min, supernatant removed, and the cell pellet resuspended in 100 µL Picopure lysis buffer (Applied Biosystems). Lysates were stored at -80°C until shipment.
100 ng of each RNA sample were processed first using a mixture of random and oligo(dT) primers and reverse transcription to generate double stranded cDNA using the Ovation Universal RNA-Seq System (NuGen, San Carlos, CA). This was followed by cDNA fragmentation to an average of 300 bp using a Covaris-2 sonicator, and then a brief S1 nuclease digestion. After purification, the cDNA was processed further through the Ovation Universal RNA-Seq System (NuGen) for end repair, barcoding, InDA-C mediated ribosomal RNA depletion, and final library production with the addition of unique nucleotide barcodes to each library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Vehicle control TSCs, repl 1
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| Data processing |
Illumina Real Time Analysis (RTA) v2.7.6
HISAT 2.0.5 alignment to MacaM v7 genome
Trimming 8 bases from 5' end of unaligned reads (from first alignment); alignment of trimmed reads
Merging accepted_hits.bam from two alignment step (samtools merge)
Removing "ExAmp" duplicates with distance threshold 2500 using proprietary Python code
Cuffdiff 2.2.1 comparisons of 6 Vehicle libraries to each group of toxicant treated libraries (5 groups, 3-6 libraries per group)
Genome_build: MacaM v7 (Macaca Mulatta)
Supplementary_files_format_and_content: gene_exp.diff files (one for each toxicant) produced by Cuffdiff
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| Submission date |
Aug 24, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Uros Midic |
| E-mail(s) |
uros@msu.edu
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| Organization name |
Michigan State University
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| Department |
Animal Sciences
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| Lab |
Latham lab
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| Street address |
474 S. Shaw Lane
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| City |
East Lansing |
| State/province |
MI |
| ZIP/Postal code |
48824-1225 |
| Country |
USA |
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| Platform ID |
GPL23949 |
| Series (1) |
| GSE103033 |
Changes in gene expression following long-term in vitro exposure of Macaca mulatta trophoblast stem cells to biologically relevant levels of endocrine disruptors |
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| Relations |
| BioSample |
SAMN07551939 |
| SRA |
SRX3124459 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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