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| Status |
Public on Sep 28, 2011 |
| Title |
Cbf1_ChIP_NoPi |
| Sample type |
SRA |
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| Source name |
yeast cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: W303
variant: cbf1::CBF1-C-Avitag-TRP1 ura3::pRS306-birA
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| Treatment protocol |
To induce the Pi starvation response, yeast cells were first grown in 10 mM Pi medium to early/mid-logarithmic phase. Cells were then harvested by filtering and washed 2 - 3 times with no Pi medium pre-warmed to 30 °C. Finally, cells were resuspended in pre-warmed no Pi medium and grown at 30 °C for 1 hour.
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| Growth protocol |
Phosphate-free synthetic complete medium was prepared from Difco phosphate-free Yeast Nitrogen Base and supplemented to final concentration of 2% glucose, 1.5 mg/ml potassium chloride, 0.1 mg/ml sodium chloride and amino acids, as described previously (Lam et al., 2008). Monobasic potassium phosphate was added to phosphate-free medium to make high phosphate (Pi) medium containing a final concentration of 10 mM inorganic phosphate. All media were adjusted to pH 4.0 with HCl (Thomas and O'Shea, 2005). Yeast strains were grown at 30 °C with shaking and cell samples were collected at early/mid-logarithmic phase (OD600 0.3 ~ 0.4).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
~100 OD units of cells were collected for high Pi and no Pi conditions. Cells were crosslinked with 1% formaldehyde for 15 minutes and then quenched with 125 mM glycine for 5 minutes at room temperature. Cells were collected by centrifugation, immediately washed with cold PBS buffer (137 mM NaCl, 2.7 mM KCl, 10 mM Sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4), and mechanically lysed with glass beads in lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate). Chromatin was fragmented by sonication of the lysate and the supernatant was pre-cleared with protein G dynabeads (Invitrogen) for 2 hours at 4 °C. M-280 dynabeads (Invitrogen) were blocked with lysis buffer containing 1% cold fish skin gelatin (Sigma-Aldrich) at 4 °C for 2 hours and then incubated with pre-cleared cell lysate overnight at 4°C. After incubation, the dynabeads were washed with lysis buffer, high salt wash buffer (50 mM HEPES, pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate), lithium wash buffer (10 mM Tris/HCl, pH 8.0, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.1% Na-Deoxycholate), and SDS wash buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA, 3% SDS) for 2 - 2 minutes at room temperature, and 1- 2 minutes with TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA). Crosslinks were reversed by incubation of samples at 65 °C for at least 6 hours in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.8% SDS. RNA and proteins in the samples were digested with 20 ug/ml RNase at 37 °C for 2 hours and 0.2 mg/ml proteinase K for 2 hours at 55 °C. DNA was then purified with phenol-chloroform extraction and precipitated with ethanol. 5% of the volume of cell lysate was removed after sonication and used to prepare the input DNA for each ChIP experiment. ChIP DNA concentration was estimated with the Pico-green DNA detection kit (Invitrogen). Sequencing libraries were prepared following the Illumina protocol and libraries with size between 200 to 300 bp were selected for PCR amplification and assayed with an Agilent bioanalyzer.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
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| Data processing |
Alignment: Sequence reads were obtained and mapped to the S. cerevisiae (2003) genomes using the Illumina Genome Analyzer Pipeline. All reads uniquely mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 80 bp to create summary windows.
Paired-end Data: the average insert size for WT_NoPi_Nucleosome is 154 bp, with standard deviation of 22.8 bp
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| Submission date |
May 24, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Xu Zhou |
| E-mail(s) |
[email protected]
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| Organization name |
Boston Children's Hospital
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| Department |
Pediatrics
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| Lab |
Xu Zhou
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| Street address |
300 Longwood Ave
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL9134 |
| Series (1) |
| GSE29506 |
Integrated approaches reveal determinants of genome-wide binding and function of the transcription factor Pho4 |
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| Relations |
| SRA |
SRX065607 |
| BioSample |
SAMN00620793 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM730521_Cbf1_ChIP_NoPi.wig.gz |
27.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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