|
Status |
Public on Mar 11, 2016 |
Title |
Rrm3-Flag WT |
Sample type |
genomic |
|
|
Channel 1 |
Source name |
Rrm3-Flag ChIP DNA from WRBb-9B, asynchronous culture
|
Organism |
Saccharomyces cerevisiae |
Characteristics |
strain: WRBb-9B+ pRS413-GAL::YRA1 genotype: MATa ade2-1 can1-100 trp1-1 his3-11,15 leu2-3,112 ura3::URA3/GPD-Tk(7x) Rrm3::FLAG + pRS413-GAL::YRA1 cell cycle stage: asynchronous
|
Growth protocol |
Yeast cells were grown in minimal medium + Glucose 2% to mid-log phase at 30°C. In samples with Rrm3-Flag ChIP, yeast cells were grown minimal medium + Galactose 2%
|
Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP was carried out as previously described (Katou et al. 2003, 2006): We disrupted 1.5 × 107 cells by Multi-beads shocker (MB400U, Yasui Kikai) using glass beads. Anti-HA polyclonal antibody (Abcam) was used for ChIP. Anti-FLAG M2 monoclonal (Sigma-Aldrich)
|
Label |
biotin
|
Label protocol |
Chromatin-immunprecipitated DNA was purified and amplified by random priming as described (Katou et al. 2003): A total of 5 μg of amplified DNA was digested with DNaseI to a mean size of 100 bp and purified, and the fragments were end-labeled with biotin-N6-ddATP.
|
|
|
Channel 2 |
Source name |
Input DNA from WRBb-9B
|
Organism |
Saccharomyces cerevisiae |
Characteristics |
strain: WRBb-9B+ pRS413-GAL::YRA1 genotype: MATa ade2-1 can1-100 trp1-1 his3-11,15 leu2-3,112 ura3::URA3/GPD-Tk(7x) Rrm3::FLAG + pRS413-GAL::YRA1 cell cycle stage: asynchronous
|
Growth protocol |
Yeast cells were grown in minimal medium + Glucose 2% to mid-log phase at 30°C. In samples with Rrm3-Flag ChIP, yeast cells were grown minimal medium + Galactose 2%
|
Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP was carried out as previously described (Katou et al. 2003, 2006): We disrupted 1.5 × 107 cells by Multi-beads shocker (MB400U, Yasui Kikai) using glass beads. Anti-HA polyclonal antibody (Abcam) was used for ChIP. Anti-FLAG M2 monoclonal (Sigma-Aldrich)
|
Label |
biotin
|
Label protocol |
Chromatin-immunprecipitated DNA was purified and amplified by random priming as described (Katou et al. 2003): A total of 5 μg of amplified DNA was digested with DNaseI to a mean size of 100 bp and purified, and the fragments were end-labeled with biotin-N6-ddATP.
|
|
|
|
Hybridization protocol |
Approximately 5 μg of DNA was hybridzed per array using the Affymetrix hybridization kit. The arrays were hybridized for 16 hours at 45° at 60 rpm using an Affymetrix hybridization oven. The probe was removed from these arrays and reused to hybridize the remaining three arrays.
|
Scan protocol |
Arrays were scanned on an Affymetrix Scanner 3000 7G
|
Description |
GAL::YRA1 Rrm3 exp1 and exp2
|
Data processing |
Primary data analyses were carried out using the Affymetrix TAS 1.1.02 software to obtain hybridization intensity, signal log2 ratio value and change P-value for each locus. For the discrimination of positive and negative signals for the binding, we compared the chromatin-immunprecipitated fraction with the supernatant fraction by using two criteria. First, the reliability of binding ratio was judged by change P-value (P ≤ 0.01). Second, clusters consisting of at least twenty-five contiguous probe-sets (positive signal, max. gap 250bp, min run. 100bp) that filled the above criteria were selected. Locus identification, raw signal intensity, detection signal log ratio, and change p-value was obtained using the Affymetrix TAS 1.1.02 software.
|
|
|
Submission date |
May 04, 2015 |
Last update date |
Mar 12, 2016 |
Contact name |
Andrés Aguilera |
Organization name |
CABIMER, Universidad de Sevilla
|
Department |
Genome BIology
|
Lab |
Genome Instability & Cancer
|
Street address |
Av. Américo Vespucio 24
|
City |
Seville |
ZIP/Postal code |
41092 |
Country |
Spain |
|
|
Platform ID |
GPL7250 |
Series (2) |
GSE68486 |
ChIP-chip data of HA-Yra1 and Rrm3-Flag in wild-type and cells overexpressing YRA1 |
GSE68488 |
YRA1 overexpression microarray and ChIP-chip |
|