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Status |
Public on Jun 12, 2015 |
Title |
T10_TFIIS4 |
Sample type |
RNA |
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Source name |
T10 time point after TFIIS4 inactivation
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Organism |
Paramecium tetraurelia |
Characteristics |
strain description: 51 new mt8 ∆A ∆ND7 strain: mating type VIII caryonide: monocaryonide medium: Escherichia coli WGP assay physiological state: autogamy competent
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Treatment protocol |
At each time point, 200-600 mL of Paramecium cells were centrifuged at 200 x g for 1 min and total RNA was extracted from unwashed cell pellets.
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Growth protocol |
Silencing media were prepared basically as described in (Galvani and Sperling, 2001) and (Nowak et al., 2011), by inoculating precultures of the appropriate bacterial strains into WGP (wheat grass powder - Pines International Co., Lawrence, USA) medium containing 0.1 mg/mL ampicillin. Following 6–8 hrs of shaking at 37°C, bacterial cultures were diluted six-fold into the same medium containing 0.4 mM IPTG to induce dsRNA synthesis. After overnight induction at 37°C, all silencing media were supplemented with 0.8 µg/mL β-sitosterol (Merck) before use. Paramecium cells were maintained at 27°C during vegetative growth and starvation. Cell growth was monitored by manual counting of cells in 1 mL aliquots of the culture. During starvation, the progression of autogamy through the different stages of nuclear reorganization was monitored by DAPI staining of cells.
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Extracted molecule |
total RNA |
Extraction protocol |
For each sample, total RNA was extracted from ~400,000 Paramecium cells using Trizol.
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Label |
Cy3
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Label protocol |
cDNA synthesis and labeling with Cyanine 3: 300ng of total RNA was used to synthesize cDNA according to the Sigma WGA1 TransPlex Whole Transcriptome Amplification kit protocol.
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Hybridization protocol |
Hybridization by NimbleGen company
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Scan protocol |
Scanning by PartnerChip company (MS200 Roche-NimbleGen)
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Description |
mating type VIII is a strain carrying an injection-induced macronuclear deletion of the surface antigen A gene and a silencing-induced macronuclear deletion of the ND7 gene Population of cells 10 hours after T0 with 100% of autogamy (3% fragmented macs, 97% visible anlagen)
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Data processing |
Images are treated with NimbleScan software from Nimblegen to obtain raw intensities (.RMA.calls) that were then used for analysis with GeneSpring GX11 software.
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Submission date |
Jan 05, 2015 |
Last update date |
Jun 13, 2015 |
Contact name |
Olivier ARNAIZ |
Organization name |
CNRS
|
Lab |
I2BC
|
Street address |
avenue de la terrasse
|
City |
Gif-sur-Yvette |
ZIP/Postal code |
91198 |
Country |
France |
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Platform ID |
GPL19603 |
Series (1) |
GSE64682 |
Transcriptome analysis upon TFIIS4 silencing during autogamy time course |
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