|
| Status |
Public on Nov 16, 2016 |
| Title |
SCC13 cells control replicate_LPS 4 |
| Sample type |
RNA |
| |
|
| Source name |
control LPS
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SCC13
transfection agent: pUNO
treatment: LPS 10μg/ml replicate 4
|
| Treatment protocol |
SCC13 tumor cells were stably transfected with control (pUNO, Invivogen) and TLR4 expressing plasmid (pUNO-TLR4GFP, Invivogen) following the manufacturer’s protocol. For the selection of positive clones the transfected cells were further grown in DMEM medium containing blasticidine (10μg/ml) as a selection marker. Afterwards cells were treated with 10μg/ml ultrapure LPS (Sigma) for 24 hours.
|
| Growth protocol |
SCC13 cell lines were grown in conventional DMEM medium supplied with 10% fetal calf serum
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol (Invitrogen) according to manufacturer's protocol
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng of total RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA).Quantification and and quality assessment were done using Nanodrop spectrophotometer (Witec) and Agilent 2100 Bioanalyzer (Agilent Technologies) respectively.
|
| |
|
| Hybridization protocol |
600 ng of the obtained Cy3-labeled cRNA was was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Human Sure Print GE 8x60 K v2 Microarrays (Agilent Technologies) (G4112A) 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.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent G2565CA Microarray Scanner System (Scan Control Software 8.5, Agilent) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green and Green PMT is set to 100%)
|
| Description |
gene expression after 24 hours
SCC13 skin tumor cell line stably transfected
|
| Data processing |
Normalized Signal Intensity
|
| |
|
| Submission date |
Nov 15, 2016 |
| Last update date |
Apr 23, 2018 |
| Contact name |
Guergana Iotzova-Weiss |
| E-mail(s) |
geri.weiss@web.de
|
| Organization name |
Dermatology Clinic Zurich
|
| Street address |
Gloriastrasse 31
|
| City |
Zurich |
| ZIP/Postal code |
8091 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL16699 |
| Series (1) |
| GSE89856 |
Differential gene expression profile of SCC13 tumor cells after TLR4 overexpression and LPS treatment |
|
| Relations |
| Reanalyzed by |
GSE113533 |
