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Status |
Public on Jun 01, 2012 |
Title |
LX2_culture_rep3 |
Sample type |
RNA |
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|
Source name |
LX2 cultured alone, Replicate 3
|
Organism |
Homo sapiens |
Characteristics |
cell line: LX2
|
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.
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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).
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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.
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Description |
Cell line described in Gut. 2005 Jan;54(1):142-51. PMID: 15591520.
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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).
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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. |
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