|
| Status |
Public on May 01, 2012 |
| Title |
S.cerevisiae FZF1 allele at t=0min replicate 1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Scer FZF1 t=0min
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
fzf1 allele: Scer FZF1
time: 0min
|
| Treatment protocol |
After 3 hours growth in YPD+TA, each culture was sampled for t=0. Sodium sulfite was immediately added to the cultures to a final concentration of 1mM. A second sample was taken 15 minutes after sulfite addition t=15min.
|
| Growth protocol |
Strains were grown overnight in YPD + 75mM L-tartaric acid buffered to pH 3.5 (YPD +TA). Strains were resuspended in fresh YPD + TA at an OD600 of 0.25 for a total of 100mL. Cultures were grown at 30 degrees Celcius at 200rpm.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated using Qiagen's Rnaeasy Mini Kit, following the manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
First strand cDNA on 10ug total RNA is generated by oligo-dT primed reverse transcription utilizing the Superscript Plus Indirect labeling system (Invitrogen) according to manufacturer's instructions. First-strand cDNA is purified with Zymo Research Clean and Concentrator 5 clean-up columns according to manufacturer’s instructions. First-strand cDNA is dried down to a volume of 3ul and coupled with the appropriate dye (Alexa Fluor 555 or 647) according to the Superscript Plus Indirect labeling system (Invitrogen) protocol. Coupling reactions are quenched with 3M sodium acetate (20ul). Fluorescently labeled cDNAs are purified with Zymo Research Clean and Concentrator 5 clean-up columns according to manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
reference pool made up of all samples
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
fzf1 allele: All alleles
time: All times
|
| Treatment protocol |
After 3 hours growth in YPD+TA, each culture was sampled for t=0. Sodium sulfite was immediately added to the cultures to a final concentration of 1mM. A second sample was taken 15 minutes after sulfite addition t=15min.
|
| Growth protocol |
Strains were grown overnight in YPD + 75mM L-tartaric acid buffered to pH 3.5 (YPD +TA). Strains were resuspended in fresh YPD + TA at an OD600 of 0.25 for a total of 100mL. Cultures were grown at 30 degrees Celcius at 200rpm.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
RNA was isolated using Qiagen's Rnaeasy Mini Kit, following the manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
First strand cDNA on 10ug total RNA is generated by oligo-dT primed reverse transcription utilizing the Superscript Plus Indirect labeling system (Invitrogen) according to manufacturer's instructions. First-strand cDNA is purified with Zymo Research Clean and Concentrator 5 clean-up columns according to manufacturer’s instructions. First-strand cDNA is dried down to a volume of 3ul and coupled with the appropriate dye (Alexa Fluor 555 or 647) according to the Superscript Plus Indirect labeling system (Invitrogen) protocol. Coupling reactions are quenched with 3M sodium acetate (20ul). Fluorescently labeled cDNAs are purified with Zymo Research Clean and Concentrator 5 clean-up columns according to manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
cDNA are paired and balanced by mass and mixed with hybridization buffer (Agilent 2X Gene Expression buffer, Agilent 10X Blocking agent, water) and applied to arrays. Hybridization is carried out at 65C for 20 hours. Washing procedures follow Agilent gene expression protocols.
|
| Scan protocol |
Scanned on an Axon 4000B scanner. Gridding and analysis of images is performed using Genepix v6.1 (Axon)
|
| Data processing |
BioConductor marray package in R was used for log transformation (log2 ((sample median foreground - sample median background)/(reference pool median foreground - reference pool median background))) followed by global median normalization
|
| |
|
| Submission date |
Jan 24, 2012 |
| Last update date |
May 01, 2012 |
| Contact name |
Justin Fay |
| E-mail(s) |
jfay@genetics.wustl.edu
|
| Organization name |
Washington University
|
| Street address |
Center for Genome Sciences Rm 5526, 4444 Forest Park Pkwy
|
| City |
Saint Louis |
| State/province |
Missouri |
| ZIP/Postal code |
63108 |
| Country |
USA |
| |
|
| Platform ID |
GPL9825 |
| Series (1) |
| GSE35308 |
Transcriptional response of FZF1 alleles from S. cerevisiae and S. paradoxus to the addition of sulfite |
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