|
| Status |
Public on May 21, 2009 |
| Title |
H2O2_30min_msn2/msn4/rpd3-1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
BYmsn2/msn4/rpd3 grown at 30C (time 0 reference) #1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain BY4741-msn2:KanMX/msn4::URA3/rpd3::LEU2 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 msn2::KanMX msn4::URA3 rpd3::LEU2)
|
| Treatment protocol |
Stresses were added directly to the culture at the indicated concentrations. For heat shock experiments, cells were collected by rapid filtration at 25C and resuspended in fresh YPD heated to 37C. For reverse heat shock experiments, cells were grown 3 doublings in YPD at 37C, collected by brief centrifugation, and resuspended in fresh YPD at 25C
|
| Growth protocol |
Unless otherwise noted, cells were grown 3 doublings in YPD or specified medium at 30C to OD600 ~0.5
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hot-phenol RNA extraction as described in Gasch AP Methods in Enzymology 350: 393-414
|
| Label |
Oyster 550 cyanine dye
|
| Label protocol |
Indirect labeling using amino-allyl-dUTP and cyanine dyes from Flownamics (Madison, Wi) as described in Gasch AP Methods in Enzymology 350: 393-414
|
| |
|
| Channel 2 |
| Source name |
BYmsn2/msn4/rpd3 30 min after addition of 0.4 mM H2O2 #1
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
Strain BY4741-msn2:KanMX/msn4::URA3/rpd3::LEU2 (MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 msn2::KanMX msn4::URA3 rpd3::LEU2)
|
| Treatment protocol |
Stresses were added directly to the culture at the indicated concentrations. For heat shock experiments, cells were collected by rapid filtration at 25C and resuspended in fresh YPD heated to 37C. For reverse heat shock experiments, cells were grown 3 doublings in YPD at 37C, collected by brief centrifugation, and resuspended in fresh YPD at 25C
|
| Growth protocol |
Unless otherwise noted, cells were grown 3 doublings in YPD or specified medium at 30C to OD600 ~0.5
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hot-phenol RNA extraction as described in Gasch AP Methods in Enzymology 350: 393-414
|
| Label |
Oyster cyanine 650 dye
|
| Label protocol |
Indirect labeling using amino-allyl-dUTP and cyanine dyes from Flownamics (Madison, Wi) as described in Gasch AP Methods in Enzymology 350: 393-414
|
| |
|
| |
| Hybridization protocol |
As indicated in Gasch AP Methods in Enzymology 350: 393-414
To prepare the probe for hybridization to a ~22 x 22 mm microarray, combine 5/zl of each of the Cy-dUTP labeled samples or 10/zl of the combined aminoallyllabeled sample, 1/zl of 10 mg/ml poly(A) carrier (Sigma), 2.3/zl 20× SSC, and 0.36 tzl 10% SDS for a final volume of 14/zl (if the microarray is larger than 22 x 22 mm add to the 10 #1 of probe solution 2.6 #1 poly(A), 5.0 20x SSC, and 0.78 #1 10% SSD and adjust the final volume to 30 #1 with water). Denature the probe by heating at 94 ° for 3 min, then cool to room temperature by rapidly transferring to a microfuge and spinning for 10 sec (avoid adding bubbles during handling). Do not chill the probe on ice as salt will precipitate from the solution. Prepare the hybridization chamber (Coming): place the microarray into the chamber and add ~20/zl 3× SSC to each of the hybridization wells in the chamber and an additional -~30/zl of 3x SSC divided into isolated drops on the edges of the microarray. Insufficient volume will result in inadequate chamber hydration during hybridization, causing the probe to dry out under the coverslip. Select multiple microscope slide coverslips of the appropriate size (Coming) that appear free of dust, chips, and scratches, and prop them for easy access with a tweezers. Adding the probe to the microarray and sealing with the coverslip requires some experience and personal technique. It is advisable to practice the following procedure before hand. To the center of the microarray, deposit 12-15/zl of the denatured and cooled probe for 22 x 22 mm arrays, or 28-30 #1 probe for 22 x 44 mm microarrays. Bubbles can prevent proper hybridization, and therefore they should be avoided and removed when possible. Large bubbles can be popped with the comer of a coverslip, but great care should be taken not to touch the microarray surface. Quickly place the coverslip directly over the deposited probe and gently drop or place the coverslip over the array. The coverslip should not be moved so as to prevent scratching of the microarray surface; however, in unavoidable cases it can be gently positioned by sliding. Close the hybridization chamber and gently and quickly submerge the chamber in a 65 ° water bath. For optimal signal, hybridize the arrays for 7-16 hr. For consistent results, the hybridization conditions, including hybridization time, should be maintained for all microarrays.
|
| Scan protocol |
Arrays were scanned on an Axon 4000B scanner
|
| Description |
Comparison of msn2/msn4/rpd3 before and 30 min after addition of 0.4 mM H2O2 - replicate #1
|
| Data processing |
Data were extracted using GenePix 6.0. Data were normalized by regional mean-centering as described by Lyne et al. 2003 BMC Genomics
|
| |
|
| Submission date |
Sep 20, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Audrey P. Gasch |
| E-mail(s) |
[email protected]
|
| Phone |
(608)265-0859
|
| Fax |
(608)262-2976
|
| Organization name |
University of Wisconsin Madison
|
| Department |
Laboratory of Genetics
|
| Lab |
Gasch Lab
|
| Street address |
425-g Henry Mall
|
| City |
Madison |
| State/province |
WI |
| ZIP/Postal code |
53706 |
| Country |
USA |
| |
|
| Platform ID |
GPL5915 |
| Series (1) |
| GSE9108 |
The Histone Deacetylase Rpd3p Coordinates Transient Changes in Genomic Expression in Response to Acute Stress |
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