|
| Status |
Public on Jul 22, 2013 |
| Title |
Gcn5 enrichment after recovery |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
ChIP_DNA_Gcn5_after_recovery
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
chip antibody: Gcn5
chip antibody manufacturer: Sigma
chip antibody catalog #: M5546
treatment: After stress (KCL) recovery
genetic background: BQS1350 (Rosaleny LE etc. Biol 2007, 8(6):R119)
|
| Treatment protocol |
Cells grow to log phase at a density of 1 × 107 to 2 × 107 cells/ml (sample collection A: normal growth), then and dilute to 5X106 cells/ml and subjected to stress condition by adding a equal amount of YPD medium containing 2 M KCl, making a final concentration of 1 M KCl with cell number around 2.5X106 cells/ml. (sample collection B 1hr after medium change B: during stress adaptation). When cell numbers reach to a concentration to 1X107 cells/ml (sample collected C: after stress adaptation). Cells are spanned down and medium is replaced with YPD without KCl for stress recovery with a start concentration around 2.5X106 cells/ml (Sample collection D after 1hr of medium changes: during stress recovery). After 2-3 generations, final samples are collected.(sample collection E: after recovery) . Cell number is counted every hour. Replicates are cultured as different batches.
|
| Growth protocol |
Gcn5-myc tagged strain is from (ref) and is used for stress adaptation and recovery model. The cells are cultivated at 30°C in YPD medium (1% yeast extract, 2% bacto peptone and 2% glucose)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Extraction and Chromatin IP procedures refers to Johnsson etc. EMBO reports (2009) 10, 1009. acH3K18 (ab1191) and acH4K16(ab61240) and Histone3 antibody are from Abcam and are used with 1:100 dilution for Chromatin Immuno-precipitation experiment, Gcn5-myc are from Sigma (M5546) and 1:50 dilution are used in ChIP experiment.
|
| Label |
biotin
|
| Label protocol |
According to the Affymetrix manufacturer's manual.
|
| |
|
| Channel 2 |
| Source name |
Input
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
control: input control
genetic background: BQS1350 (Rosaleny LE etc. Biol 2007, 8(6):R119)
|
| Treatment protocol |
Cells grow to log phase at a density of 1 × 107 to 2 × 107 cells/ml (sample collection A: normal growth), then and dilute to 5X106 cells/ml and subjected to stress condition by adding a equal amount of YPD medium containing 2 M KCl, making a final concentration of 1 M KCl with cell number around 2.5X106 cells/ml. (sample collection B 1hr after medium change B: during stress adaptation). When cell numbers reach to a concentration to 1X107 cells/ml (sample collected C: after stress adaptation). Cells are spanned down and medium is replaced with YPD without KCl for stress recovery with a start concentration around 2.5X106 cells/ml (Sample collection D after 1hr of medium changes: during stress recovery). After 2-3 generations, final samples are collected.(sample collection E: after recovery) . Cell number is counted every hour. Replicates are cultured as different batches.
|
| Growth protocol |
Gcn5-myc tagged strain is from (ref) and is used for stress adaptation and recovery model. The cells are cultivated at 30°C in YPD medium (1% yeast extract, 2% bacto peptone and 2% glucose)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Extraction and Chromatin IP procedures refers to Johnsson etc. EMBO reports (2009) 10, 1009. acH3K18 (ab1191) and acH4K16(ab61240) and Histone3 antibody are from Abcam and are used with 1:100 dilution for Chromatin Immuno-precipitation experiment, Gcn5-myc are from Sigma (M5546) and 1:50 dilution are used in ChIP experiment.
|
| Label |
biotin
|
| Label protocol |
According to the Affymetrix manufacturer's manual.
|
| |
|
| |
| Hybridization protocol |
According to the Affymetrix manufacturer's manual.
|
| Scan protocol |
According to the Affymetrix manufacturer's manual.
|
| Description |
Gcn5 enrichment after stress recovery compared to input control
|
| Data processing |
The file is processed from the bar files. Gcn5 enrichment at normal conditions is provided for each gene in separate files. Each genes are provided with 38 data points, with the first 9 points show the equal bins of 5'IGR region (start s from the milddle of upstream intergenic regions and end before the starting code), 20 equal bins of ORF region, and 9 equal bins of 3'IGR region (starts after the stop code and end at the middle of the intergenic region).
Raw data from Affymetrix (CEL format) were analyzed using Tiling Analysis software (TAS) from affymetrix to find the enrichment for whole genome regions (bar file). The data for the 5 conditions are normalized to the have the same medium value. Average gene analysis was done using a procedure modified from Pokholok et al (2005) and Li et al (2007). Briefly, the ORF region of each gene in S. cerevisiae was divided into 20 equally sized bins. while the of upstream 5´IGR region starts from the milddle of upstream intergenic regions and end before the starting code and and downstream 3ÍGR region starts after the stop code and end at the middle of the intergenic region. Both 5’IGR and 3’IGR are divided in to 9 equal sized bins. The java code for average gene analysis is in the supplementary filesX and with a 2-clause BSD license.
|
| |
|
| Submission date |
Mar 19, 2012 |
| Last update date |
Nov 15, 2014 |
| Contact name |
Anthony Wright |
| E-mail(s) |
yongtao.xue.franzen@ki.se
|
| Organization name |
Karolinska Institute
|
| Lab |
Anthony Wright
|
| Street address |
Blickagången 6
|
| City |
Huddinge |
| ZIP/Postal code |
141 57 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL7250 |
| Series (2) |
| GSE36600 |
Genome-wide enrichment of Gcn5 and H3K18/H4K16 acetylation under physiological change of stress adaptation and stress recovery |
| GSE36601 |
Yeast under physiological changes of stress adaptation and stress recovery |
|
