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Status |
Public on Jan 20, 2015 |
Title |
SEN_cDNA2_exp1 |
Sample type |
RNA |
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Source name |
WI38 hTERT RAF1-ER cells treated with 4-HT for 3 days to induce senescence
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Organism |
Homo sapiens |
Characteristics |
cell type: WI38 hTERT RAF1-ER
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Treatment protocol |
For senescence induction, cells were treated with 20 nM 4-hydroxy-tamoxifen (4-HT) (Sigma) for 3 days.
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Growth protocol |
WI38 hTERT RAF1-ER cells were maintained in MEM supplemented with glutamine, non-essential amino acids, sodium pyruvate, penicillin-streptomycin, and 10% FBS as per the ATCC in normoxic (5 % O2) culture conditions.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA were prepared using the MasterPure RNA Purification Kit (Epicentre Biotechnologies) supplemented with Baseline-ZERO DNase (Epicentre)
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Label |
biotin
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Label protocol |
Two different cDNAs populations were produced from the total RNA. cDNA1 was synthesized from 250 ng of total RNA (without rRNA reduction) according to the “GeneChip Whole Transcript Sense Target Labeling Assay” (Affymetrix) and is identical to RNA. cDNA2 was synthesized from 12 microgr of total RNA, as previously, but starting from the second cycle of cDNA synthesis thus skipping the first cycle cDNA synthesis and the “In Vitro Transcription” amplification step. Therefore, cDNA2 is complementary to RNA.
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Hybridization protocol |
Differential cDNA populations were hybridized on the GeneChip Human Tiling 2.0R-A arrays (Affymetrix), which contain DNA probes from the forward DNA strand. Hybridization and scanning were performed according to the manufacturer protocol at the Plateforme Biopuces (INSA, Toulouse). Plus and minus strand expression were monitored by the cDNA2 and cDNA1 experiments, respectively.
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Scan protocol |
Tiling arrays were scanned with a 7000 3G Affymetrix scanner with autoloader and analyzed with Tiling Analysis Software (TAS)
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Data processing |
Scanned array data normalization (quantile normalization, scale to 500) as well as calculation of the log2 (ratio) of the senescence signal over the control signal were performed using TAS (Affymetrix).
The result files are:CDNA1SENVSCONT-EXP1_signalSL.chp, CDNA2SENVSCONT-EXP1_signalSL.chp, CDNA1SENVSCONT_EXP2_signalSL.chp, CDNA2SENVSCONT_EXP2_signalSL.chp.
For images, data were calculated using a bandwidth of 300 (cDNA1SENvsCONT_EXP1_signal300.chp, cDNA2SENvsCONT_EXP1_signal300.chp, cDNA1SENvsCONT_EXP2_signal300.chp, cDNA2SENvsCONT_EXP2_signal300.chp) and visualized using the Integrated Genome Browser (IGB, Affymetrix) genome version H_sapiens_Mar_2006.
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Submission date |
Dec 22, 2013 |
Last update date |
Jan 20, 2015 |
Contact name |
Estelle Nicolas |
E-mail(s) |
estelle.nicolas@univ-tlse3.fr
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Phone |
+33 5 61 55 81 11
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Organization name |
INSERM
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Lab |
UMR5077, MCD, CBI
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Street address |
118 route de Narbonne
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City |
Toulouse |
ZIP/Postal code |
31062 |
Country |
France |
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Platform ID |
GPL4910 |
Series (1) |
GSE53578 |
A vlincRNA participates in senescence maintenance by relieving H2AZ mediated repression at the INK4 locus |
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