|
| Status |
Public on Mar 13, 2023 |
| Title |
High stiffness replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
MCF10CA1a
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10CA1a
stiffness: High stiffness (8000 Pa)
|
| Treatment protocol |
MCF10CA1a cells were cultured for five days on low (400 Pa) or high (5000 Pa) 50 mm polyacrylamide-based hydrogels. Gels were prepared in house and were conjugated with growth factor-reduced Matrigel (0.14 mg/ml) prior to cell plating.
|
| Growth protocol |
Cells were maintained in DMEM/F12 (Gibco) media supplemented with 5% horse serum (Gibco), 10 mM HEPES (Gibco) and 0.029 M sodium bicarbonate (Gibco).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The coverslip gels were removed from the culture dishes and edges were carefully wiped clean with a cotton swab. Cells were then lysed by placing the coverslip gel-side down in 500 µl Lysis solution/2-Mercaptoethanol mixture from the GenElute™ mammalian total RNA miniprep kit (Sigma-Aldrich) and RNA was isolated according to the manufacturer’s protocol.
|
| Label |
biotin
|
| Label protocol |
A total of 200 ng of RNA was used to generate amplified sense strand DNA targets with Ambion WT Expression Kit followed by fragmentation and biotinylation with Affymetrix GeneChip WT Terminal Labeling Kit.
|
| |
|
| Hybridization protocol |
2.5 ug of ssDNA target was then hybridized to Human Gene 1.0 ST Array in a GeneChip Hybridization Oven 645 (16h-18 h at 45° C).
|
| Scan protocol |
The arrays were washed and stained in a Gene Chip Fluidics Station 450 and finally scanned in an Affymetrix Gene Chip Scanner 3000 7G.
|
| Data processing |
DNA-microarray raw data (.cel files) were summarized to gene level expression values using the Oligo package. A custom cdf based on the entrez gene data base (version 25.0.0) was used as it provides improved precision and accuracy. Following normalization using the robust microarray average (RMA) method, 20513 unique genes were included.
|
| |
|
| Submission date |
Jun 10, 2022 |
| Last update date |
Mar 13, 2023 |
| Contact name |
Ola Larsson |
| E-mail(s) |
ola.larsson@ki.se
|
| Phone |
+46 (0)8 517 73280
|
| Organization name |
Karolinska Institutet
|
| Department |
Department of oncology-pathology
|
| Street address |
CCK, R8:01
|
| City |
Stockholm |
| ZIP/Postal code |
171 76 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL32334 |
| Series (2) |
| GSE205817 |
Transcriptomic profiling of breast cancer cells on hydrogels with different stiffness [array] |
| GSE205819 |
Transcriptomic profiling of breast cancer cells on hydrogels with different stiffness |
|
