|
| Status |
Public on Feb 25, 2019 |
| Title |
Matrigel, UC supnat |
| Sample type |
RNA |
| |
|
| Source name |
fibroblasts, 18h, UC supnat control
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: fibroblasts
cell source: ATCC-1459
|
| Treatment protocol |
Medium from organoid cultures containing 7x105 cells or from empty Matrigel droplets were centrifuged at 300 g for 5 min and 2,000 g for 15 min to remove cells and cell debris, respectively. The supernatnant was then ultracentrifuged at 4C, 100,000 g for 70 min. The EV-enriched pellet was re-suspended in the supernatant and fibroblasts were treated with the EV-containing pellet or EV-free supernatant.
|
| Growth protocol |
Fibroblasts were grown in DMEM 4500 g/l glucose, Pen/Strept, glutamine and 5% EV-free FBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNEasy micro Kit (Qiagen), according to the manufacturer's protocol with on-column Dnase digestion.
|
| Label |
Cy3
|
| Label protocol |
200 ng total RNA was reverse transcribed and processed according to Low Input QuickAmp version 6.5. cRNA was purified on Qiagen RNEasy columns.
|
| |
|
| Hybridization protocol |
1650 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutesfollowing the manufacturers instructions. On completion of the fragmentation reaction, 55ul 2x Gex HiRPM hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome 4 x44K Microarrays (G2519F) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent), 1 minute with 37 °C GE Wash buffer 2 (Agilent) and then in Agilent Drying Solution.
|
| Scan protocol |
Slides were scanned immediately after washing on Agilent B Scanner, Extended Dynamic Range (100% and 10% laser intensity, 5 micron resolution)
|
| Description |
Medium from 3D culture only with Matrigel was ultracentrifuged and fibroblasts were treated with the supernatant without Evs
|
| Data processing |
Feature Extraction Software 12.0.3.1., default one-color high density protocol (GE1_1200_Jun14). Data were imported into Chipster (https://chipster.csc.fi/) and normalized with the default Agilent 1-color protocol in Chipster (background treatment: normexp, background offset 50, normalize chips: quantile, remove control probes: yes)
|
| |
|
| Submission date |
May 29, 2018 |
| Last update date |
Feb 25, 2019 |
| Contact name |
Zoltan Wiener |
| E-mail(s) |
[email protected]
|
| Organization name |
Semmelweis University
|
| Street address |
Ulloi ut 26
|
| City |
Budapest |
| ZIP/Postal code |
H-1085 |
| Country |
Hungary |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE114979 |
The effect of colorectal cancer (CRC) organoid-derived extracellular vesicles on the expression profile of fibroblasts |
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