|
| Status |
Public on Mar 17, 2020 |
| Title |
wt_30_M.w1001 |
| Sample type |
genomic |
| |
|
| Source name |
WT
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: WT
treatment: Untreated
cell-cycle phase: S-Phase 30min
antibody: anti-BrDU
strain: W303-872
|
| Treatment protocol |
Samples were released from alpha-factor arrest in the presence or not of HU (200mM) and collected at the indicated time post G1 release
|
| Growth protocol |
Cells were grown in YPD at 25°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Standard protocol for BrdU-IP chip. For immunoprecipitation of BrdU-labeled DNA, 1.5 × 10e9 cells were lysed five times for 2 min each time in NIB buffer (17% (v/v) glycerol, 50 mM MOPS buffer, 150 mM potassium acetate, 2 mM magnesium chloride, 500 μM spermidine and 150 μM spermine, pH 7.2) with zirconium beads on a Vibrax shaker (VXR basic, Ika) at 4 °C. DNA was isolated using Qiagen genomic DNA extraction kit and sonicated to yield an average DNA size of 300–600 bp. For each immunoprecipitation, 10 μl of mouse anti-BrdU IgG1 (BD Bioscience, 555627) was prebound to Dynabeads M-280 sheep anti-mouse IgG (Invitrogen, 112.01D) and added to the denatured purified DNA.
|
| Label |
Biotin
|
| Label protocol |
Purified immunoprecipitated DNA was amplified using the WGA2 GenomePlex Complete Whole Genome Amplification kit (Sigma) with the addition of 0.1 mM dUTP in the amplification reaction. Amplified DNA (7 μg) was fragmented and labeled using the GeneChip WT Double-Stranded DNA Terminal Labeling kit (Affymetrix, PN 900812) according to the manufacturer's recommendations.
|
| |
|
| Hybridization protocol |
DNA was hybridized to Affymetrix GeneChip S. cerevisiae Tiling 1.0R Array (5-bp resolution) using the GeneChip Hybridization, Wash, and Stain kit (Affymetrix, PN 900720).
|
| Scan protocol |
Tiling arrays were scanned with the GeneChip scanner 3000 7G.
|
| Description |
Ip-BrdU of Wild type 30' after release in S-Phase
|
| Data processing |
Signals were normalized with TAS 1.1.02 (Affymetrix) using quantile normalization. Analysis results were stored in log2 scale for signal intensity and in −10 × log10 scale for P values. Probe signals were analyzed using perfect matches only with a bandwidth of 300 bp. P-value intervals were generated using the following parameters: cutoff, 10−5; maximum gap, 80 bp; min run, 40 bp. Significant intervals were scored as active origins when signal intensity was above a threshold arbitrarily set at 50% of the signal range and when BrdU tracks were larger than 1 kb. Replication profiles (signal log ratio) were displayed with IGB 6.1 Bar files correspond to signal intensities (signal log ratio of BrdU IP relative to WCE) and can be displayed with IGB (Integrated genome Brower).
|
| |
|
| Submission date |
Aug 29, 2019 |
| Last update date |
Mar 18, 2020 |
| Contact name |
romain forey |
| E-mail(s) |
romain.forey@epfl.ch
|
| Organization name |
IGH
|
| Street address |
142 avenue de la Cardonille
|
| City |
Montpellier |
| ZIP/Postal code |
34000 |
| Country |
France |
| |
|
| Platform ID |
GPL7250 |
| Series (2) |
| GSE136601 |
Mec1 is activated at the onset of normal S phase by low dNTP pools impeding DNA replication [microarray] |
| GSE136605 |
Mec1 is activated at the onset of normal S phase by low dNTP pools impeding DNA replication |
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