|
| Status |
Public on Sep 06, 2022 |
| Title |
HepaRG, day 34, 4 days DMSO removed, replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
HepaRG, day 34, 4 days DMSO removed, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepaRG
cell type: hepatic stem cell
day: 34
dmso: yes (day 15 to day 30) and no (day30 to day 34)
|
| Growth protocol |
Cells were seeded at a density of 2.7 x 104/cm2 and maintained for two weeks in a William’s E medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, 5 μg/ml insulin, and 50 μM hydrocortisone. Then, the culture medium was or not supplemented with 2% DMSO for two additional weeks. Cells were usually collected at different stages of differentiation process: D4, D15, D30 - DMSO and D30 + DMSO. However, to determine the influence of DMSO, it was removed from differentiated HepaRG culture (D30 + DMSO) for further 4 days and cells collected at 34 days (D34 +/- DMSO)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA extractions were performed with RNeasy mini kit (Qiagen) following the manufacturer's recommendations. RNA was qualified using a Nanodrop 2000 spectrophotometer (Thermo Fischer Scientific, Waltham, MA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the One-Color Low RNA Input Quick Amp Labelling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop (Thermo Fischer Scientific, Waltham, MA). amplification yield was 4.04 ± 0.53 µg cRNA and specific activity was 11.97 ± 0.98 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 µl 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 Scanner (G2565) using one color scan setting for 8x60k array slides (profile Agilent G3-GX-1color).
|
| Description |
Gene expression of HepaRG cells at day 34. DMSO removed from differentiated HepaRG culture (D30 + DMSO) for further 4 days
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using protocol GE_1-107_sep09_ssSurogates and Grid: 028004_D_F_20110325. Gene expression data were further analyzed by using the GeneSpring software (Agilent Technologies). Filtration by flag and signal intensity was applied. Briefly, were retained only the entities in which at least 100% of the values in at least one condition had a detected flag (i.e. a positive and significant feature as defined by GeneSpring). For the filtration by signal intensity, were retained the entities in which 100% of the values in at least one condition were within the range of interest (i.e. 20-100th percentile).
|
| |
|
| Submission date |
Mar 21, 2018 |
| Last update date |
Sep 06, 2022 |
| Contact name |
Hélène Dubois-Pot-Schneider |
| E-mail(s) |
helene.dubois-pot-schneider@univ-lorraine.fr
|
| Organization name |
Université de Lorraine - UMR7039 CRAN
|
| Department |
CRAN
|
| Lab |
Département Biologie, Signaux et Système en Cancérologie et Neurosciences (BioSiS)
|
| Street address |
Site CRAN/Faculté de Médecine - Bâtiment D, 1er étage 9 avenue de la Forêt de Haye, BP 184
|
| City |
Vandœuvre-lès-Nancy |
| ZIP/Postal code |
54505 |
| Country |
France |
| |
|
| Platform ID |
GPL14550 |
| Series (1) |
| GSE112123 |
DMSO effect on HepaRG differentiation |
|
