|
| Status |
Public on Oct 20, 2008 |
| Title |
H1.3 sh -dox repl.1 |
| Sample type |
RNA |
| |
|
| Source name |
Histone H1.3 shRNA vector; uninduced, no doxycyclin treatment
|
| Organism |
Homo sapiens |
| Characteristics |
T47D-MTVL (breast cancer cell line carrying one stably integrated copy of luciferase reporter gene driven by the MMTV promoter) stably infected with an inducible system for the expression of shRNAs.Cells stably express RedFP and KRAB repressor fused to Tet regulator.Upon Dox treatment, cells express RedFP and the cloned shRNA.
no depletion
Histone H1.3 shRNA vector
uninduced, no doxycyclin treatment
|
| Treatment protocol |
Plasmids for the lentivirus vector-mediated drug-inducible RNA interference system (pLVTHM, ptTR-KRAB-Red, pCMC-R8.91 and pMD.G) were provided by D. Trono (University of Geneva). The 64-mer oligonucleotides for histone H1 shRNA cloning into MluI/ClaI-digested pLVTHM were designed, annealed and phosphorylated as communicated by D. Trono (http://tronolab.epfl.ch/). Oligonucleotides have the following general structure: 5'-CGCGTCCCC-N19 TTCAAGAGA-rcN19-TTTTTGGAAAT-3' and 5'-AGGGG-N19-AAGTTCTCT-rcN19-AAAAACCTTTAGC-3', being N19 the specific target sequence for each H1 variant and rcN19 its reverse complementary sequence. The corresponding 19-mer gene-specific target sequences for interference were designed manually following standard rules (AAN19, GC % 30-70). Target sequences (N19) are CGCTGACTCGCAGATCAAG for H1.0, AGAGCGTAGCGGAGTTTCT for H1.2, CTGCCAAGAGTCCAGCTAA for H1.3, GAAGAGCGCCAAGAAGACC for H1.4 and GGCAACTAAGAAGGCTGCC for H1.5. For the production of viral particles containing the HIV-derived vectors, 2.5x106 HEK 293T cells (Clontech) were transfected with plasmids ptTR-KRAB-Red, pLVTHM-shH1.n or pEV833-HA-H1.n (10µg), pCMV-R8.91 (6.5µg) and pMD.G (3.5µg) in 10 cm dishes using calcium phosphate. Medium was collected every 24 hours for 2 days and centrifuged 1h30min at 26.000 rpm at 4ºC in a sucrose gradient to concentrate viruses. Pellet containing viral particles was dissolved in medium and used for cell infection. Cells were infected using the spinoculation method, i.e. plates were centrifuged at 1,200 x g for 2 hr at 25 ºC. 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 the RNAsy Kit (Qiagen). RNA concentration was measured with a small volume spectrophotometer (Nanodrop). The quality of the RNA was analyzed using the Agilent Bioanalyzer 2100 and the RNA 6000 LabChip Kit (Agilent) with the Eukaryote Total RNA Nano Assay, with RIN ranging between 9.2 and 9.8.
|
| Label |
biotin / streptavidin-Cy3
|
| Label protocol |
For each sample 200 ng of total RNA were reverse transcribed, amplified by in vitro transcription and labelled with biotin-UTP using the Illumina Total Prep RNA amplification kit (IL1791, Ambion) following the manufacturer's instructions. Labelled sample quality was assessed by spectrophotometry and bioanalyzer. After preheating at 65ºC for 5 min.
|
| |
|
| Hybridization protocol |
For each sample, 750 ng of biotinylated cRNA were hybridized in a BeadChip Hyb Chamber with rocking 16 h at 58ºC. The day after the bead arrays were washed with Illumina proprietary washing solutions in a Hybex waterbath: first with static incubation for 10 min at 55ºC in E1BC solution, followed by 10 rinses by dipping in the same solution and shaking 5 min at 90 rpm in an orbital shaker; the next wash was by dipping 10 times in 100% ethanol and shaking 10 min at 110 rpm in an orbital shaker; this was followed by another wash in E1BC solution with 10 dippings followed by 2 min shaking at 90 rpm. Washed bead arrays were blocked in E1 buffer 10 min in rocking incubator and 10 additional minutes with 2 ml of E1 buffer plus streptavidin-Cy3. The fluorescent reagent was washed away with E1BC solution with 10 dippings plus 5 min shaking at 140 rpm. Finally beadarrays were dried by centrifugation 4 min at 275 rcf, followed by scanning in a Illumina Beadstation.
|
| Scan protocol |
The Beadscan software was used to control de scanner, generate .tif images and extract the raw data as tabulated text files. Default settings for DirectHyb Gene Expression were used.
|
| Description |
Negative control for histone H1.3 RNAi, biological replicate 1 of 2; T47D-MTVL cells transfected with Dox-responsive KRAB repressor and RedFP (ptTR-KRAB-Red) and H1.3 shRNA expression vector (pLVTHM) uninduced
|
| Data processing |
The raw data was summarized per probe using BeadStudio software Gene Expression module and the summary data file was processed using the PILLA web einterface tool (Lozano et al, unpublished), an implementation of the Lumi package (Du et al, 2008) developed within the Bioconductor project in the R statistical programming environment (Gentleman et al, 2004). Data were normalized using the rsn method and vst as the variance stabilization method. The log2 intensities were median centered and log ratios were computed as differences in log2 intensities for each probe. The SAM (significance analysis of microarrays) two class unpaired comparison test was applied with 100 permutations to detect statistically significant differences in gene expression between treated and control conditions (Tusher et al, 2001).
|
| |
|
| Submission date |
Jul 30, 2008 |
| Last update date |
Oct 20, 2008 |
| Contact name |
Lauro Sumoy |
| E-mail(s) |
[email protected]
|
| 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 |
GPL6104 |
| Series (1) |
| GSE12299 |
Gene expression in response to depletion of specific human histone H1 isoforms. |
|
