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| Status |
Public on Mar 13, 2017 |
| Title |
022_PCSK9-C_10uM_EGF |
| Sample type |
SRA |
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| Source name |
PTEC-TERT1_PCSK9-C_10uM_EGF
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| Organism |
Homo sapiens |
| Characteristics |
cell type: PTEC-TERT1 cells
antisense oligonucleotides (asos): PCSK9-C
concentration: 10uM
exogeneous egf: TRUE
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| Treatment protocol |
For ASO toxicity assessment, PTEC-TERT1 were seeded into 96-well plates (Falcon, 353219) at a density of 40 000 and 20 000 cells/well respectively in PTEC medium and grown until confluence prior to treatment with ASOs. ASOs were dissolved in PBS and added to the cell culture at a final concentration of 10, 30 or 100 µM in a final volume of 100 µl. Medium was changed, stored at -20ᵒC for cytokine analysis and refreshed along with AONs every 3 days. PBS served as vehicle control.
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| Growth protocol |
PTEC-TERT1 (Evercyte GmbH, Austria) were cultured according to the manufacturer’s instructions in PTEC medium [DMEM/F12 without phenol red (Invitrogen 110390-021) containing 1% Pen/Strep (Gibco: 15140-122), 10 mM Hepes (Gibco: 15630-056) , 5.0 µg/ml human insulin (Gibco: 41400-045), 5.0 µg/ml human transferrin (Gibco: 41400-045), 8.65 ng/ml sodium selenite (Gibco: 41400-045), 0.1µM hydrocortisone (Sigma H6909), 10 ng/ml human recombinant Epidermal Growth Factor (R&D Systems : 236-EG-200), 3.5µg/ml ascorbic acid (SIGMA : A4544 powder), 25 ng/ml prostaglandin E1(SIGMA : P5515), 3.2 pg/ml Triiodo-L-thyronine (Sigma T-5516) and 100 µg/ml Geneticin (Gibco: 10131-027)].
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cell were harvested 6h after ASO addition by removing the culture medium. Cell lysates in 50 µl RLT buffer (QIAGEN, Hombrechtikon, Switzerland) were immediately frozen and stored at -80°C.
10 ng of total RNA from each time point and biological replicate was reverse transcribed to cDNA and amplified by limited PCR according to the protocol supplied with the Ion AmpliSeq™ RNA Library Kit (Life Technologies, Carlsbad, USA, Catalog number 4482335). After primer digestion, adapters and barcodes were ligated to the amplicons followed by magnetic bead purification. The purified library was amplified, purified and stored at -20°C. Amplicon size and DNA concentration was measured using an Agilent High Sensitivity DNA Kit (Agilent Technologies, Waldbronn, Germany) according to the manufacturer's guide.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent PGM |
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| Description |
PTEC-TERT1 cells treated with PCSK9-C (10uM) for 6h in the presence of exogeneous EGF
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| Data processing |
Base-calling, filtering, QC, and alignment was handled automatically by IonTorrent Server Suite (version 4.0.2)
Amplicon coverage is reported by IonTorrent Server Suite (version 4.0.2) as a tab-delimited file, which was converted into GCT format by in-house scripts.
Genome_build: hg19
Supplementary_files_format_and_content: GCT format, with amplicon counts for pathway reporter genes in each sample
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| Submission date |
Jun 15, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Jitao David Zhang |
| E-mail(s) |
jitao_david.zhang@roche.com
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| Phone |
+41616886251
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| Organization name |
F. Hoffmann-La Roche
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| Department |
Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel
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| Lab |
Pharmaceutical Sciences
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| Street address |
Grenzacherstrasse 124
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| City |
Basel |
| ZIP/Postal code |
4070 |
| Country |
Switzerland |
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| Platform ID |
GPL17301 |
| Series (1) |
| GSE83392 |
Molecular phenotyping of antisense oligonucleotides (ASOs) in immortalized proximal tubule epithelial cells (PTEC-TERT1) |
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| Relations |
| BioSample |
SAMN05253925 |
| SRA |
SRX1847328 |
| 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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