|
| Status |
Public on Oct 29, 2010 |
| Title |
br1_untreated_cu_tr1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
CuSO4
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/background: BY4741a
genotype: pdr5Δ
statin treated: no
cuso4 treated: yes
znso4 treated: no
|
| Treatment protocol |
Cultures were spun down and resuspended in fresh media (SC, 2% glucose, 0.25 mM EDTA) either with or without statin (5 μg/mL) at an OD600 of 0.1. Metabolites were added as indicated and the vehicle (DMSO) concentration was adjusted to 0.1% (v/v) in all cases. The yeast were harvested at approximately 50% of the eventual maximum OD600 (~0.4 for +statin, ~0.8 for –statin).
|
| Growth protocol |
Yeast (BY4741a) carrying the pdr5Δ mutation was grown to saturation in SC media + 2% glucose for 20-24 hr at 30°C with constant agitation. The culture was back diluted to 0.01 OD600 (-statin) or 0.09 OD600 (+statin, 5 μg/mL), and these pretreatment cultures were grown for an additional 18-20 hr, at which point an OD600 of approximately 0.4-0.6 was reached.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were collected using a 0.45 μm vacuum filter with nylon filter membranes and the cell cake was rapidly frozen in liquid nitrogen and stored at -80 °C. RNA isolation was initiated by adding 4 mL lysis buffer (0.1 M EDTA, 0.5% SDS, 0.01 M Tris pH 7.5) to the frozen filter membrane and vortexing thoroughly. Then, 3 mL acid phenol was added and the tube was vortexed and placed in a 65 °C water bath for 1 hr with additional vortexing every 20 min. The filter paper was removed with tweezers and the liquid poured into a phase lock gel tube (5Prime, Gaithersburg, GA), incubated at 4 °C for 10 min and centrifuged for 10 min at 1880 RCF. 3 mL of chloroform was added, and the tube was inverted 3 times to mix and then centrifuged at 1880 RCF for 10 min. The chloroform extraction was repeated and the aqueous layer was poured into a new 15 mL conical tube. The RNA was precipitated by adding 1/10 volume 3 M sodium acetate and 2 volumes of 100% ethanol followed by mixing and incubation overnight at -20 °C. The precipitated RNA was pelleted and washed twice with 70% ethanol, then reconstituted in 250 μL deionized, RNAse-free H2O.
|
| Label |
Cy3
|
| Label protocol |
For dye labeling, 5 μg of RNA were purified using the Qiagen RNeasy kit (Qiagen, Valencia, CA), and a reference RNA sample was created by pooling RNA from all 6 conditions. RNA was labeled following the Agilent two-color gene expression protocol v5.7 (Agilent Technologies, Santa Clara, CA), although only 25% of the recommended amounts of Cy3 and Cy5 dyes were used in each reaction.
|
| |
|
| Channel 2 |
| Source name |
Reference RNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain/background: BY4741a
genotype: pdr5Δ
composition: mix of RNA from all 6 experimental conditions
|
| Treatment protocol |
Cultures were spun down and resuspended in fresh media (SC, 2% glucose, 0.25 mM EDTA) either with or without statin (5 μg/mL) at an OD600 of 0.1. Metabolites were added as indicated and the vehicle (DMSO) concentration was adjusted to 0.1% (v/v) in all cases. The yeast were harvested at approximately 50% of the eventual maximum OD600 (~0.4 for +statin, ~0.8 for –statin).
|
| Growth protocol |
Yeast (BY4741a) carrying the pdr5Δ mutation was grown to saturation in SC media + 2% glucose for 20-24 hr at 30°C with constant agitation. The culture was back diluted to 0.01 OD600 (-statin) or 0.09 OD600 (+statin, 5 μg/mL), and these pretreatment cultures were grown for an additional 18-20 hr, at which point an OD600 of approximately 0.4-0.6 was reached.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cultures were collected using a 0.45 μm vacuum filter with nylon filter membranes and the cell cake was rapidly frozen in liquid nitrogen and stored at -80 °C. RNA isolation was initiated by adding 4 mL lysis buffer (0.1 M EDTA, 0.5% SDS, 0.01 M Tris pH 7.5) to the frozen filter membrane and vortexing thoroughly. Then, 3 mL acid phenol was added and the tube was vortexed and placed in a 65 °C water bath for 1 hr with additional vortexing every 20 min. The filter paper was removed with tweezers and the liquid poured into a phase lock gel tube (5Prime, Gaithersburg, GA), incubated at 4 °C for 10 min and centrifuged for 10 min at 1880 RCF. 3 mL of chloroform was added, and the tube was inverted 3 times to mix and then centrifuged at 1880 RCF for 10 min. The chloroform extraction was repeated and the aqueous layer was poured into a new 15 mL conical tube. The RNA was precipitated by adding 1/10 volume 3 M sodium acetate and 2 volumes of 100% ethanol followed by mixing and incubation overnight at -20 °C. The precipitated RNA was pelleted and washed twice with 70% ethanol, then reconstituted in 250 μL deionized, RNAse-free H2O.
|
| Label |
Cy5
|
| Label protocol |
For dye labeling, 5 μg of RNA were purified using the Qiagen RNeasy kit (Qiagen, Valencia, CA), and a reference RNA sample was created by pooling RNA from all 6 conditions. RNA was labeled following the Agilent two-color gene expression protocol v5.7 (Agilent Technologies, Santa Clara, CA), although only 25% of the recommended amounts of Cy3 and Cy5 dyes were used in each reaction.
|
| |
|
| |
| Hybridization protocol |
RNA was hybridized to Agilent S. cerevisiae v2 (8 x 15k) microarrays following the Agilent two-color gene expression protocol v5.7 (Agilent Technologies, Santa Clara, CA). Slides were washed according to the Agilent protocol. There were two randomly chosen conditions that were replicated per slide; these technical replicates were used to assess RNA labeling and hybridization quality. Three independent biological replicates were performed according to the procedure outlined above.
|
| Scan protocol |
Slides were scanned on an Agilent G2505C scanner. Images were quantified using Agilent Feature Extraction Software (version 10.5.1.1).
|
| Description |
Biological replicate 1.
Technical replicate 1.
|
| Data processing |
Agilent Feature Extraction Software (version 10.5.1.1) was used for background subtraction and LOWESS normalization.
|
| |
|
| Submission date |
Oct 28, 2010 |
| Last update date |
Jan 23, 2015 |
| Contact name |
Douglas McKay Fowler |
| Organization name |
University of Washington
|
| Department |
Genome Sciences
|
| Lab |
Stanley Fields
|
| Street address |
1705 NE Pacific St, Foege Building S313
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98195 |
| Country |
USA |
| |
|
| Platform ID |
GPL9825 |
| Series (1) |
| GSE25004 |
Suppression of statin effectiveness by copper and zinc in yeast and human cells |
|
