|
| Status |
Public on May 05, 2014 |
| Title |
400uM Glutamine Pulse t=300 min |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
yeast nitrogen-limited chemostat
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
nutrient limitation or pulse: 400uM Glutamine
steady-state or pulse: pulse
|
| Treatment protocol |
All nitrogen-limiting media contained 800µM nitrogen regardless of the molecular form of the nitrogen and 1 g/L CaCl2-2H2O, 1 g/L of NaCl, 5 g/L of MgSO4-7H2O, 10 g/L KH2PO4, 2% glucose and trace metals and vitamins
|
| Growth protocol |
Chemostat cultures were maintained in aerobic Sixfors fermentors (Infors) at 30°C using constant stirring at 400rpm
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
mRNA was prepared using hot acid phenol exatraction followed by purification using a QIAGEN RNeasy mini kit
|
| Label |
Cy5
|
| Label protocol |
cDNA from each sample was generated using MMLV reverse transcriptase primed with a poly-dT primer containing the promoter sequence for T7 RNA polymerase. Labeled cRNA was generated using T7 RNA polymerase and UTPs labeled with either Cy3 or Cy5.
|
| |
|
| Channel 2 |
| Source name |
Ammonium limited chemostat D=0.12
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
nutrient limitation: ammonium
steady-state or pulse: steady-state
|
| Treatment protocol |
All nitrogen-limiting media contained 800µM nitrogen regardless of the molecular form of the nitrogen and 1 g/L CaCl2-2H2O, 1 g/L of NaCl, 5 g/L of MgSO4-7H2O, 10 g/L KH2PO4, 2% glucose and trace metals and vitamins
|
| Growth protocol |
Chemostat cultures were maintained in aerobic Sixfors fermentors (Infors) at 30°C using constant stirring at 400rpm
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
mRNA was prepared using hot acid phenol exatraction followed by purification using a QIAGEN RNeasy mini kit
|
| Label |
Cy3
|
| Label protocol |
cDNA from each sample was generated using MMLV reverse transcriptase primed with a poly-dT primer containing the promoter sequence for T7 RNA polymerase. Labeled cRNA was generated using T7 RNA polymerase and UTPs labeled with either Cy3 or Cy5.
|
| |
|
| |
| Hybridization protocol |
Cy3 and Cy5 labeled cRNA was co-hybridized to an Agilent 60-mer DNA microarray at 65C for 16 hours. DNA microarrays were washed using the standard Agilent wash protocol.
|
| Scan protocol |
DNA microarrays were scanned using an Agilent Technologies Scanner G2505B
|
| Description |
gene expression in nitrogen limited chemostats
|
| Data processing |
Probe intensities were determined using the Agilent Feature Exractor software and normalized between the Cy5 and Cy3 channel using a linear-loess normalization
|
| |
|
| Submission date |
May 05, 2014 |
| Last update date |
May 05, 2014 |
| Contact name |
David Gresham |
| E-mail(s) |
dgresham@nyu.edu
|
| Organization name |
New York University
|
| Department |
Biology
|
| Lab |
Gresham Lab
|
| Street address |
12 Waverly Place, Rm 203
|
| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10003 |
| Country |
USA |
| |
|
| Platform ID |
GPL9294 |
| Series (1) |
| GSE57293 |
Dynamics of Nitrogen-regulated Gene Expression Reveals a Reciprocal Relationship between Cell Growth Rate and Nitrogen Catabolism |
|
