|
| Status |
Public on Apr 05, 2015 |
| Title |
AGM static 36hrs rep3 |
| Sample type |
RNA |
| |
|
| Source name |
AGM static 36hrs rep3
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
developmental stage: E10.5
tissue: AGM
duration of fluid flow: 36 hours
force: 0.0001 dyne/cm^2
drug: vehicle
|
| Treatment protocol |
Microfluidics consisted of IBIDI VI^0.4 channel slides in line with a recirculating medium system driven by a single Harvard Apparatus PHD ULTRA 4400 with Remote syringe pump, 0.27 PSI crack pressure check valves (Qosina), and female luer lock three-way stop cocks (Qosina). After 6 to 8 hours of incubation, non-adherent cells were washed away and cells were cultured for 6 or 36 hours in static/low-flow conditions (0.0001 dyne/cm^2) or in the presence of WSS (5 dyne/cm^2).
|
| Growth protocol |
Embryos from C57BL/6J timed pregnancies were microdissected for isolation of E10.5 AGM regions. Tissues were dissociated by treatment with Accutase (STEMCELL Technologies) at room temperature with gentle agitation for 20 minutes. Cells were resuspended in M5300 Myelocult medium (STEMCELL Technologies) enriched with HEPES (Invitrogen, 12.5 ml 1M HEPES/500 ml of Myelocult medium), non-Essential Amino Acids (Gibco), Sodium Pyruvate (Gibco), Penicillin 10 units/ml (Lonza) and Streptomycin 10 µg/ml (Lonza) without addition of hydrocortisone.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were directly lysed on microfluidic slides with RLT lysis buffer. Total RNA was immediately extracted with the QIAGEN RNeasy kit.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared with the Ambion MessageAmp kit for Illumina arrays
|
| |
|
| Hybridization protocol |
Standard Illumina hybridization protocol
|
| Scan protocol |
Standard Illumina scanning protocol
|
| Description |
-
|
| Data processing |
Raw signals of all the build-in controls were checked as quality control for the performance of the arrays. Sample-independent controls were used to check: a. Hybridization (control molecules at low, medium and high concentrations); b. Signal generation (background, noise, biotin labeling and hybridization at high and low stringency). Housekeeping genes were used as sample-dependent controls. Background was subtracted and arrays were quantile normalized.
|
| |
|
| Submission date |
Oct 17, 2014 |
| Last update date |
Apr 05, 2015 |
| Contact name |
Pamela L Wenzel |
| Organization name |
University of Texas Health Science Center at Houston
|
| Department |
Integrative Biology & Pharmacology
|
| Lab |
Pamela Wenzel
|
| Street address |
6431 Fannin St, MSB 4.130
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL17543 |
| Series (1) |
| GSE62463 |
Effects of shear stress on cells of the aorta-gonad-mesonephros (AGM) |
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