|
| Status |
Public on Jun 01, 2012 |
| Title |
HepaRG_coculture_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
HepaRG cocultured with LX2, Replicate 3
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepaRG
|
| Growth protocol |
HepaRG and LX2 cells were grown in serum- and DMSO-free William's E medium using 6-well plates. For coculture experiments, 1 µm pore size transwell inserts which allow diffusion of media components but prevent cell migration (BD Biosciences, San Jose, CA) were used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified using the RNAeasy kit (Qiagen, Valencia, CA, USA) following the manufacturer’s recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was amplified and labeled with Cy3 fluorescent dye using Agilent one color low-input QuickAmp labeling kit following according to the manufacturer's instructions. Starting from 150 ng total RNA, amplification yield was 9.7±0.6 μg cRNA and specific activity was 20.3±1.3 pmol Cy3 per μg cRNA.
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 µ of 2x Agilent hybridization buffer (HI-RPM) was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K 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) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray C Scanner (G2565) using one color scan setting for 8x60k array slides.
|
| Description |
Cell line described in Proc Natl Acad Sci U S A. 2002 Nov 26;99(24):15655-60. PMID: 12432097.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09_ssSurrogates and Grid: 028004_D_F_20100430).
|
| |
|
| Submission date |
Oct 03, 2011 |
| Last update date |
Jun 01, 2012 |
| Contact name |
Cedric Coulouarn |
| E-mail(s) |
cedric.coulouarn@inserm.fr
|
| Organization name |
INSERM
|
| Department |
U1242
|
| Lab |
COSS
|
| Street address |
CLCC Eugène Marquis, Rue de la Bataille Flandres Dunkerque, Bat D, 1er étage
|
| City |
Rennes |
| ZIP/Postal code |
35042 |
| Country |
France |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE32565 |
Molecular crosstalk between hepatocytes and hepatic stellate cells. |
|
