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| Status |
Public on May 16, 2015 |
| Title |
Control siRNA, 15 dynes/cm2 shear stress. |
| Sample type |
SRA |
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| Source name |
aortic valve endothelial cells
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| Organism |
Homo sapiens |
| Characteristics |
treatment: Control siRNA, 15 dynes/cm2 shear stress
cell type: Primary aortic valve endothelial cells
passage: p8
primary source: 55 yr old female (heart transplant pulmonary hypertension)
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| Treatment protocol |
siRNAs were delivered into endothelial cells using RNAiMax (Life Technologies, Carlsbad, CA). The siRNAs used were NOTCH1 siRNA (cat# 4392422 s963)and control scrambled siRNA (cat# 4390843) (Ambion/Life Technologies). Endothelial cell media was changed to optimem with no suplements (Gibco/Life Technologies), siRNA/RNAiMax was added for 4 hours. Then, the media was changed to full ECM and the cells were harvested for analysis 48 hours later.
Cells were cultured in “flow” or pulsatile shear stress conditions using microSlide I 0.8 Luer channel slides and flow kit (Ibidi, Verona, WI) and a L/S® Modular brushless digital dispensing drive peristaltic pump with 6-roller cartridge pump head (Cole Parmer, Vernon Hills, IL). Shear stress conditions were between 13 and 15 dynes/cm2.
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| Growth protocol |
Cells were grown on plates coated with 10 mg/mL fibronectin from bovine plasma (Sigma, St Loius, MO) and grown in endothelial cell media (ECM)(Sciencell) at 5% CO2. Media was changed every other day and cells were passaged with 0.05% Trypsin EDTA when confluent.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using trizol extraction followed by RNeasy MinElute Cleanup Kit with on column DNAse digestion (Qiagen, Valencia, CA).
Biological triplicates were pooled prior to library generation. 50 ng of RNA was used with the Ovation® RNA-Seq System (7100-08)(NuGEN, San Carlos, CA) and manufacturer’s protocol to synthesize and SPIA amplify cDNA, which was then sheared with a covaris S-series. After end repair, purification and P1 P2 adaptor ligation, templated beads were generated with the EZ beads system, and libraries where then paired end sequened (50bp forward, 35bp reverse) on a SOLiD4 DNA sequencing system (Life Technologies).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB SOLiD 4 System |
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| Data processing |
RNA-seq reads were aligned to the human hg19 genome using TopHat
TopHat was supplied with GTF-format annotation of known exon positions. Novel exon discovery was disallowed—only exons and splice junctions that were already present in the annotation were included. Duplicate reads (reads mapping to the exact same start / stop position) were removed using the Picard tool MarkDuplicates
Secondary mapping locations were discarded. For each read, only the top alignment was retained.
For paired-end reads, any "singleton" reads (where both ends did not map successfully) were discarded.
Cufflinks was used to obtain a reads-per-gene total and provide normalized expression levels, reported as RPKM (reads mapped to a gene per thousand bases of the gene, per million mapped reads)
Differentially expressed genes were identified using Cuffdiff, part of the Cufflinks suite. Only genes with RPKMs > 1 and CuffDiff output of “OK” were included in subsequent analysis
Genome_build: hg19
Supplementary_files_format_and_content: Processed Data file includes gene ID, gene short name, locus, FPKM and status for each condition in tab delimeted .txt file.
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| Submission date |
May 21, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Deepak Srivastava |
| E-mail(s) |
dsrivastava@gladstone.ucsf.edu
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| Organization name |
J. David Gladstone Institutes
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| Department |
Gladstone Institute of Cardiovascular Disease
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| Lab |
Deepak Srivastava
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| Street address |
1650 Owens St.
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL13393 |
| Series (2) |
| GSE47157 |
Shear Stress and NOTCH1 Regulate Calcification Related Genes in Human Aortic Valve Endothelium (RNA-Seq) |
| GSE47160 |
Shear Stress and NOTCH1 Regulate Calcification Related Genes in Human Aortic Valve Endothelium |
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| Relations |
| BioSample |
SAMN02152544 |
| SRA |
SRX285611 |
| 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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