|
| Status |
Public on Feb 21, 2014 |
| Title |
taf9-tCRD2 SM-induced vs uninduced_4 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
YSS26
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: taf9-tCRD2
treatment: +Sulfometuron Methyl
|
| Treatment protocol |
Sulfometuron Methyl, an inhibitor of ILV2 gene product, causes isoleucine-valine starvation, and consequent induction of Gcn4. The SM treatment was done as per the Growth protocol.
|
| Growth protocol |
Yeast strains were cultured in Synthetic Complete (SC)-Leu-Ile-Val medium for 36h at 30°C, 220 rpm. Fresh cultures were inoculated in duplicate with the pre-culture, for the wild-type at an OD600 of 0.04, and for the taf9-tCRD2 strain at an OD600 of 0.08. Untreated wild-type and taf9-tCRD2 cultures were harvested at an OD600 of 0.5. For SM treatment, cultures were grown to an OD600 of 0.5 and induced with Sulfometuron Methyl (SM) for 2h at 30°C and harvested by rapid filtration on sterile Millipore 0.45mm filter disks, and frozen at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by hot-phenol extraction method as follows. Cells were washed with AE buffer and about 10 OD equivalent of cells were re-suspended in 500µl of AE buffer, 33.4µl of 25% (w/v) SDS and 500µl phenol equilibrated with AE buffer. The mixture was vortexed and incubated in a water bath set to 65°C for 10 min with vortexing every 1-2 min. The mixtures were rapidly cooled on ice and centrifuged at RT for 10 min at 13,000 rpm. The resulting aqueous phase was collected, ~0.542 ml chloroform added to the supernatant, mixed, centrifuged at RT for 10 min and the aqueous phase collected. About 1/10th volume of 3M NaOAc (pH 5.3) and an equal volume of isopropanol were added, mixed, immediately centrifuged at 13,000 rpm for 45 min at 4°C. The pellet was washed with chilled 70% ethanol and centrifuged at 13,000 rpm for 20 min at 4°C. The pellet was air-dried and re-suspended in 50µl of 0.1% (w/v) DEPC treated, autoclaved, double-distilled water, and purified using RNeasy kit from Qiagen. The RNA samples were quantitated in a Nanodrop spectrophotometer and the integrity was assessed on an agarose gel, as well as on the Agilent 2100 Bioanalyzer.
|
| Label |
cy5
|
| Label protocol |
The total RNA were labeled using the Agilent Quick Amp Kit PLUS (Part number: 5190-0424). Five hundred nanograms each of the samples were reverse transcribed at 40°C and converted to double stranded cDNA was primed using oligodT tagged with a T7 polymerase promoter. The double-stranded cDNA was used as template in an in vitro transcription reaction at 40°C to generate cRNA. The samples were labeled with Cy3-CTP or Cy5-CTP (Agilent) during this step. Labeled cRNA was cleaned up and quality assessed for yields and specific activity using Nanodrop.
|
| |
|
| Channel 2 |
| Source name |
YSS26
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: taf9-tCRD2
treatment: -Sulfometuron Methyl
|
| Treatment protocol |
Sulfometuron Methyl, an inhibitor of ILV2 gene product, causes isoleucine-valine starvation, and consequent induction of Gcn4. The SM treatment was done as per the Growth protocol.
|
| Growth protocol |
Yeast strains were cultured in Synthetic Complete (SC)-Leu-Ile-Val medium for 36h at 30°C, 220 rpm. Fresh cultures were inoculated in duplicate with the pre-culture, for the wild-type at an OD600 of 0.04, and for the taf9-tCRD2 strain at an OD600 of 0.08. Untreated wild-type and taf9-tCRD2 cultures were harvested at an OD600 of 0.5. For SM treatment, cultures were grown to an OD600 of 0.5 and induced with Sulfometuron Methyl (SM) for 2h at 30°C and harvested by rapid filtration on sterile Millipore 0.45mm filter disks, and frozen at -80°C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted by hot-phenol extraction method as follows. Cells were washed with AE buffer and about 10 OD equivalent of cells were re-suspended in 500µl of AE buffer, 33.4µl of 25% (w/v) SDS and 500µl phenol equilibrated with AE buffer. The mixture was vortexed and incubated in a water bath set to 65°C for 10 min with vortexing every 1-2 min. The mixtures were rapidly cooled on ice and centrifuged at RT for 10 min at 13,000 rpm. The resulting aqueous phase was collected, ~0.542 ml chloroform added to the supernatant, mixed, centrifuged at RT for 10 min and the aqueous phase collected. About 1/10th volume of 3M NaOAc (pH 5.3) and an equal volume of isopropanol were added, mixed, immediately centrifuged at 13,000 rpm for 45 min at 4°C. The pellet was washed with chilled 70% ethanol and centrifuged at 13,000 rpm for 20 min at 4°C. The pellet was air-dried and re-suspended in 50µl of 0.1% (w/v) DEPC treated, autoclaved, double-distilled water, and purified using RNeasy kit from Qiagen. The RNA samples were quantitated in a Nanodrop spectrophotometer and the integrity was assessed on an agarose gel, as well as on the Agilent 2100 Bioanalyzer.
|
| Label |
cy3
|
| Label protocol |
The total RNA were labeled using the Agilent Quick Amp Kit PLUS (Part number: 5190-0424). Five hundred nanograms each of the samples were reverse transcribed at 40°C and converted to double stranded cDNA was primed using oligodT tagged with a T7 polymerase promoter. The double-stranded cDNA was used as template in an in vitro transcription reaction at 40°C to generate cRNA. The samples were labeled with Cy3-CTP or Cy5-CTP (Agilent) during this step. Labeled cRNA was cleaned up and quality assessed for yields and specific activity using Nanodrop.
|
| |
|
| |
| Hybridization protocol |
The labeled cRNA samples were hybridized on to an Agilent Custom Saccharomyces cerevisiae 8x15k Gene expression Array (AMADID: 016333) designed by Genotypic Technology Private Limited. About 300ng of Cy3 labeled samples and 300ng of Cy5 labeled samples were pooled, fragmented and hybridized using the Gene Expression Hybridization kit (Agilent Part Number 5188-5242) in Agilent’s Surehyb Chambers at 65°C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327), and dried.
|
| Scan protocol |
The slides were scanned using the Agilent Microarray Scanner G2505C at 5mm resolution.
|
| Description |
Biological replicate 4 of 4. Comparison of mutant taf9-tCRD2 in SM inducing versus uninducing conditions.
|
| Data processing |
The scanned images were processed in the Feature Extraction Software v10.7.3.1, and the raw data was obtained. Data normalization was done using GeneSpring GX v11.5 (Agilent) using the recommended (Lowess) normalization.
|
| |
|
| Submission date |
Feb 21, 2013 |
| Last update date |
Feb 21, 2014 |
| Contact name |
Krishnamurthy Natarajan |
| E-mail(s) |
nat0200@mail.jnu.ac.in, nat0200@gmail.com
|
| Organization name |
Jawaharlal Nehru University
|
| Department |
School of life sciences
|
| Lab |
Laboratory of Eukaryotic Gene Regulation
|
| Street address |
314/325, School of life sciences, JNU
|
| City |
New Delhi |
| State/province |
Delhi |
| ZIP/Postal code |
110067 |
| Country |
India |
| |
|
| Platform ID |
GPL14009 |
| Series (1) |
| GSE44544 |
Evolutionarily conserved C-terminal region of TAF9 is critical for SAGA and TFIID recruitment to promoters and transcriptional activation |
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