|
| Status |
Public on Sep 02, 2011 |
| Title |
Wild Type cells |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: BMA wild-type
|
| Growth protocol |
Liquid suspension cultures were inoculated from starter cultures and grown O/N in YPAD at 30C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen, Rneasy Mini Kit cat No.74104, As per the manufacturers instructions.
|
| Label |
cy5
|
| Label protocol |
The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). Five hundred nanograms each of the Control and test samples were incubated with reverse trancription mix at 40°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields and specific activity
|
| |
|
| Channel 2 |
| Source name |
WT cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: BMA wild-type
|
| Growth protocol |
Liquid suspension cultures were inoculated from starter cultures and grown O/N in YPAD at 30C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Qiagen, Rneasy Mini Kit cat No.74104, As per the manufacturers instructions.
|
| Label |
cy3
|
| Label protocol |
The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). Five hundred nanograms each of the Control and test samples were incubated with reverse trancription mix at 40°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up and quality assessed for yields and specific activity
|
| |
|
| |
| Hybridization protocol |
300ng of cy3 labeled samples and 300ng of cy5 labeled samples were fragmented and hybridized on the slides . Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). Hybridization was carried out in Agilent’s Surehyb Chambers at 65º C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327) and scanned using the Agilent Microarray Scanner G2505C at 5 micron resolution.
|
| Scan protocol |
Scanned on an Agilent Technologies Scanner G2505C at 5 micron and Images were quantified using Agilent Feature Extraction Software (version10.7.3.1)
|
| Description |
Biological replicates of Wild type cells
|
| Data processing |
Channel split analysis was performed. Probe signal intensities generated from feature extraction raw data were split based on channel type cy3 and cy5 [BMA B_Cy5, BMA A_Cy3, 3KO B_Cy5, 3KO A_Cy3 , 1358 B_Cy5 , 1358 A_Cy3]. Data analysis performed and ratios were obtained for the following: (1) 3KO B_Cy5 vs. BMA B_Cy5 (2) 1358 B_Cy5 vs. BMA B_Cy5 (3) 3KO A_Cy3 vs. BMA A_Cy3 (4) 1358 A_Cy3 vs. BMA A_Cy3 . Python programming language was used for the normalization and analysis. Gene based analysis is performed. [BMA is yeast wild type strain as control in the analysis]
|
| |
|
| Submission date |
Sep 01, 2011 |
| Last update date |
Sep 02, 2011 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL14009 |
| Series (1) |
| GSE31833 |
A role for Snf2 related nucleosome spacing enzymes in genome-wide nucleosome organization |
|
