|
| Status |
Public on May 16, 2014 |
| Title |
WT replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
WT
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: T47D-MTVL
cell type: human breast cancer cells
genotype/variation: overexpressing HA-tagged wild type H1.4
dox treatment: Untreated
depletion/expression status: Histone H1.4 expression
|
| Treatment protocol |
Initially, a cell line expressing the Dox-responsive KRAB repressor and RedFP (ptTR-KRAB-Red) was generated. Then, this cell line was infected with viruses for expression of the different H1 variants shRNAs (pLVTHM). The inducible knocked-down cell lines were sorted in a FACSvantageSE (Becton Dickinson) for RedFP-positive and GFP-positive fluorescence after 3 days of Dox treatment. Then cells were amplified in the absence of Dox until an experiment was performed. Doxicycline (Sigma) was added at 2.5 µg/ml when indicated. Along a 6-day treatment with Dox, cells were passaged at day 3. Serum-containing media was replaced with serum-free media at day 4 for growth arrest. Cells overexpressing WT or K26A HA-tagged H1.4 were grown without treatments.
|
| Growth protocol |
Cell lines were grown in RPMI 1640 medium, supplemented with 10% FBS, 2mM L-glutamine, 100 U/ml penicillin, and 100µg/ml streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using High Pure RNA isolation kit (Roche) according to the manufacturer's instructions. cDNA was obtained from 100 ng of total RNA using SuperScript VILO cDNA synthesis (Invitrogen). High RNA integrity was assessed by Bioanalyzer nano 6000 assay with RIN numbers 9.5
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA was prepared using the Illumina Total Prep RNA Amplification Kit (Ambion, Cat #4393543)
|
| |
|
| Hybridization protocol |
cRNA was purified and hybridized on Illumina HumanRef-8 V3.0 Expression BeadChips
|
| Scan protocol |
Standard Illumina scanning protocol
|
| Description |
6_JM14
|
| Data processing |
Raw intensity data was taken from the Final Report produced by the GenomeStudio software version 1.6.0 (Illumina).
Variance‐stabilizing transformation (vst) and robust spline normalization (rsn) were applied to the raw data.
|
| |
|
| Submission date |
Mar 11, 2014 |
| Last update date |
May 16, 2014 |
| Contact name |
Albert Jordan |
| E-mail(s) |
ajvbmc@ibmb.csic.es
|
| Organization name |
IBMB-CSIC
|
| Department |
Molecular Genomics
|
| Street address |
Baldiri Reixac 4
|
| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
| |
|
| Platform ID |
GPL18405 |
| Series (1) |
| GSE55786 |
Gene expression of T47D-MTVL human breast cancer cells upon H1.4 knock-down or overexpression |
|
