|
| Status |
Public on Apr 01, 2017 |
| Title |
control LEC, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Human intestinal lymphatic endothelial cells isolated from jejunum
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: intestinal lymphatic endothelial cells
treatment: control
|
| Treatment protocol |
Cells were seeded at 30’000 cells/cm2 and transfected 18 hours later with 33 nM siRNA using Lipofectamine RNAiMAX (Invitrogen). A SMARTpool (ThermoScientific, ref.L-016083-00) comprising 4 different siRNAs, or a single independent siRNA (GGAUUCGAAUGCGCCAAGA, Ambion) were used to knockdown TAZ. AllStars Negative Control siRNA (Qiagen) was used as a control. Cells were collected 6 hours post-transfection by trypsin-treatment, and seeded at confluency (50’000 cells/cm2) on chamber slides pre-coated with 3 µg/cm2 fibronectin.
|
| Growth protocol |
Human intestinal lymphatic endothelial cells were isolated from jejunum as described (Norrmen et al, 2010, Blood). Cells were grown on plates coated with 0.5 µg/cm2 fibronectin (Roche) in EBM-2 complete medium (Lonza).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected 48h post-transfection with a 30-min trypsin treatment at 37°C, followed by trypsin inactivation by diluting 7x in complete medium. Cells were lysed in RLT buffer (Qiagen) and total RNA was extracted using the Qiagen RNeasy Micro Kit.
|
| Label |
biotin
|
| Label protocol |
For each sample, 100ng of total RNA were amplified using the WT sense strand Target Labelling kit (Affymetrix, Cat.no. 900223); 5.5μg of the resulting sense cDNA was fragmented by UDG (uracil DNA glycosylase) and APE 1 (apurinic/apyrimidic endonuclease 1) and biotin-labelled with TdT (terminal deoxynucleotidyl transferase) using the GeneChip® WT Terminal labelling kit (Affymetrix Cat.no. 900671, Santa Clara, USA).
|
| |
|
| Hybridization protocol |
Affymetrix Human Gene 1.0 ST arrays (Affymetrix, Santa Clara , CA, USA) were hybridized with 2.7μg of biotinylated target, at 45°C for 17 hours washed and stained according to the protocol described in Affymetrix GeneChip® Expression Analysis Manual (Fluidics protocol FS450_0007).
|
| Scan protocol |
The arrays were scanned using the GeneChip® Scanner 3000 7G (Affymetrix) and raw data was extracted from the scanned images and analyzed with the Affymetrix Power Tools software package (Affymetrix).
|
| Description |
Gene expression data from control human intestinal LEC
|
| Data processing |
The data was corrected for batch effect with the COMBAT method, which adjusts for known batches using an empirical Bayes framework , and was normalized using Robust Multichip Average (method Bioconductor package affy, R version 3.0.1).
|
| |
|
| Submission date |
Apr 10, 2015 |
| Last update date |
Apr 01, 2017 |
| Contact name |
Nadine Fournier |
| E-mail(s) |
nadine.fournier@sib.swiss
|
| Organization name |
Agora Cancer Research Center & Swiss Institute of Bioinformatics
|
| Lab |
Translational Data Science Facility
|
| Street address |
Agora Cancer Research Center, Bugon 25A
|
| City |
Lausanne |
| State/province |
Vaud |
| ZIP/Postal code |
1000 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE67760 |
Expression profile of human lymphatic endothelial cells in the presence or absence of TAZ |
|
