|
| Status |
Public on Apr 02, 2015 |
| Title |
Ctrl_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
breast cancer cell T47D-MTVL (breast cancer cell line carrying one stably integrated copy of luciferase reporter gene driven by the MMTV promoter)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: T47D-MTVL
gender: female
tissue: breast cancer ductal carcinoma
dox treatment: Untreated
depletion/expression: no depletion
expression: Random shRNA vector
|
| 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.
|
| 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 |
Cy3
|
| Label protocol |
For each sample 100 ng of total RNA mixed with 2 ul of 1:10000 Agilent One Color Spike Mix were reversed transcribed into cDNA with a T7 promoter and the cDNA was in vitro transcribed into cRNA in the presence of Cy3-CTP using the Low input quick Amp kit (Agilent). Labelled samples were purified using RNeasy mini spin columns (Qagen) and yield and specific activity wwere assessed by spectrophotometry (Nanodrop) and confirmed to be within specifications (>825 ng and >6 pmol Cy3/ug cRNA). 600 ng of cRNA were preblocked and fragmented in Agilent fragmentation buffer and mixed with Agilent GEx Hybridization mix.
|
| |
|
| Hybridization protocol |
Hybridization mix was laid onto each sector of subarray gasket slide and sandwiched against a 8 x 65K format oligonucleotide microarray (Human v1 Sureprint G3 Human GE 8x60k Microarray, Agilent design ID 028004) inside a hybridization chamber which wqs hybridized overnight at 65ºC in a rotisserie oven rotating at 10 rpm for a total of 17 hours. Subsequently array chambers were disassembled submerged in Agilent Gene Expression Buffer 1 and washed 1 minute in another dish with the same solution with a magnetic stirrer at 200 rpm at room temperature, followed by 1 minute in gilent Gene Expression Buffer 2 with a magnetic stirrer at 200 rpm at 37ºC and immediate withdrawal from the solution and air drying.
|
| Scan protocol |
Fluorescent signal was captured into TIF images with an Agilent scanner using recommended settings (20 bit, 5 um resolution, 100% PMT and 100% laser power) with Scan Control software (Agilent).
|
| Description |
negative control for transfection, T47D-MTVL breast cancer cell line transfected with doxicyclin inducible scrambled shRNA expression vector without doxicyclin treatment, replicate 1 of 2
|
| Data processing |
Signal intensities were extracted into a tabulated text file using Feature Extraction software (Agilent) using the appropriate array configuration and annotation files. The normalized log2intensities were obtained using quantile method with normexp background correction the Bioconductor Limma package in R.
|
| |
|
| Submission date |
Oct 28, 2014 |
| Last update date |
Apr 02, 2015 |
| Contact name |
Lauro Sumoy |
| E-mail(s) |
lsumoy@igtp.cat
|
| Organization name |
IGTP
|
| Department |
High Content Genomics and Bioinformatics
|
| Street address |
Ctra. Can Ruti, Camí de les escoles s/n
|
| City |
Badalona |
| State/province |
Barcelona |
| ZIP/Postal code |
08916 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE62766 |
Gene expression of T47D-MTVL human breast cancer cells upon H1X knock-down |
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