|
| Status |
Public on Jun 20, 2013 |
| Title |
252035410005_2_2-DS/LOG[B(3)/B(5)] |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
LOG_B
|
| Organism |
Kluyveromyces lactis |
| Characteristics |
phase: LOG
organism: Kluyveromyces lactis
|
| Treatment protocol |
Glucose depletion: Samples from the BMW cultures were taken at: Lag, Log, Late log (LL), Diauxic shift (DS), Post shift (PS), and plateau (P) at the appropriate times for each species. Samples were collected in 50 mL conicals filled with the appropriate amount of 100% methanol in a dry ice-ethanol bath to stop cellular processes and RNase activity at a temperature of – 40 ° C or lower to produce a 60/40 mixture once the sample is added. Cells were pelleted at 3000 rpm at -4°C, flash frozen with liquid N2, and stored at -80°C prior to total RNA extraction.
|
| Growth protocol |
Cells were plated onto BMW plates from frozen glycerol stocks. After 2 days, cells were taken from plates and grown overnight in BMW medium at 30 ° C in a New Brunswick Scientific Edison model TC-7 roller drum on the highest speed until saturated (1-2 days). These saturated cultures were then used to inoculate 300 ml BMW batch cultures in 2 liter Erlenmeyer flasks at 1 x 106 cells/mL for the glucose depletion and repletion experiments described below. Flasks were transferred to New Brunswick Scientific Edison water bath model C76 shakers set to 200 rpm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen Yeast RNA extraction
|
| Label |
Cy5
|
| Label protocol |
Total RNA samples were reverse transcribed to cDNA and labeled with either Cy3 or Cy5 using a modification of the protocol developed by Joe Derisi (UCSF) and Rosetta Inpharmatics (Kirkland, WA) that can be obtained at www.microarrays.com.
|
| |
|
| Channel 2 |
| Source name |
LL_B
|
| Organism |
Kluyveromyces lactis |
| Characteristics |
phase: LL
organism: Kluyveromyces lactis
|
| Treatment protocol |
Glucose depletion: Samples from the BMW cultures were taken at: Lag, Log, Late log (LL), Diauxic shift (DS), Post shift (PS), and plateau (P) at the appropriate times for each species. Samples were collected in 50 mL conicals filled with the appropriate amount of 100% methanol in a dry ice-ethanol bath to stop cellular processes and RNase activity at a temperature of – 40 ° C or lower to produce a 60/40 mixture once the sample is added. Cells were pelleted at 3000 rpm at -4°C, flash frozen with liquid N2, and stored at -80°C prior to total RNA extraction.
|
| Growth protocol |
Cells were plated onto BMW plates from frozen glycerol stocks. After 2 days, cells were taken from plates and grown overnight in BMW medium at 30 ° C in a New Brunswick Scientific Edison model TC-7 roller drum on the highest speed until saturated (1-2 days). These saturated cultures were then used to inoculate 300 ml BMW batch cultures in 2 liter Erlenmeyer flasks at 1 x 106 cells/mL for the glucose depletion and repletion experiments described below. Flasks were transferred to New Brunswick Scientific Edison water bath model C76 shakers set to 200 rpm.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen Yeast RNA extraction
|
| Label |
Cy3
|
| Label protocol |
Total RNA samples were reverse transcribed to cDNA and labeled with either Cy3 or Cy5 using a modification of the protocol developed by Joe Derisi (UCSF) and Rosetta Inpharmatics (Kirkland, WA) that can be obtained at www.microarrays.com.
|
| |
|
| |
| Hybridization protocol |
Standard Agilent protocols for Agilent 8x15K and 4x44K Oligo Microarrays
|
| Scan protocol |
Arrays were scanned using an Agilent scanner and analyzed with Agilent’s feature extraction software version 10.5.1.1.
|
| Description |
LL
Biological replicate B, technical replicate 2 (dye swap)
|
| Data processing |
Agilent Feature Extraction Software (v 10.5.1.1) was used for background subtraction and LOWESS normalization. Reported expression values for each gene are median log2 ratios across all probes.
|
| |
|
| Submission date |
Mar 03, 2012 |
| Last update date |
Jun 21, 2013 |
| Contact name |
Dawn Thompson |
| E-mail(s) |
dawnt@broadinstitute.org
|
| Organization name |
Broad Institute
|
| Lab |
Aviv Regev
|
| Street address |
7 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL10499 |
| Series (1) |
| GSE36253 |
Evolutionary principles of modular gene regulation in Yeasts |
|
