|
| Status |
Public on Jan 02, 2013 |
| Title |
ChIP Sequencing Pho7-TAP -Pi |
| Sample type |
SRA |
| |
|
| Source name |
DP94
|
| Organism |
Schizosaccharomyces pombe 972h- |
| Characteristics |
phosphate concentration: 0 mM KH2PO4
chip antibody: anti-Protein A (Sigma)
time: 2 hours post-starvation
|
| Treatment protocol |
Following growth in high-Pi media, cells were split into either 200 mL of high-Pi or no-Pi media and grown for 2 hours at 30°C, 220RPM. Formaldehyde (Sigma) was added to a final concentration of 1% (v/v) to cross-link chromatin, and the reaction was allowed to proceed for 15 minutes. Glycine (Sigma) was then added to a final concentration of 125 mM and incubated for 5 minutes to quench cross-linking. Cells were collected by centrifugation and washed once with cold PBS buffer, pH 7.4. The cell pellet was snap frozen in liquid nitrogen and stored in −80 °C.
|
| Growth protocol |
S. pombe cells were maintained in previously described YES or EMM media. Prior to starvation, cells were grown at 30°C, 220RPM to mid-log phase (OD600~0.2) in 90%SD-10%EMM media supplemented with 10 mM KH2PO4 (high-Pi media).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by bead beating (6 x 2 min on, 2 min off) and chromatin was sheared to 300-600 bp fragments using a Misonix Sonicator 3000. Immunoprecipitation was performed with 100 uL of Protein G Dynabeads (Invitrogen) coupled to 4 uL of anti-Protein A antibody (Sigma). Protein concentrations were measured using a Bradford Assay (Bio-Rad). Following the generation of ChIP lysate three aliquots of 650 μg soluble protein were subject to immunoprecipitation and pooled just prior to elution from the beads. Samples were processed following the Illumina HTS guidelines with libraries of 200-300 bp selected via 2% agarose DNA gels. Libraries were amplified by PCR and purity was determined using an Agilent High-Sensitivity DNA kit on an Agilent Bioanalyzer. Libraries were sequenced on an Illumina HighSeq 2000 using a 50 bp read length.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
ChIP Sequencing
|
| Data processing |
Sequenced reads were aligned to the S. pombe 972 h- genome using ELAND. Uniquely mappable reads with a maximum of 3 mismatches were mapped to the genome. Reads were then extended 80 base-pairs to cover the average length of insert DNA between sequencing adaptors as determined by the Agilent Bioanalyzer. WIG files for ChIP normalized such that the genome average is 100 reads/nucleotide.
Genome_build: S. pombe 972 h-
Supplementary_files_format_and_content: WIG files for each chromosome
|
| |
|
| Submission date |
Jul 19, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Ian O'Brien Carter-O'Connell |
| Organization name |
Harvard University
|
| Department |
MCB
|
| Lab |
O'Shea
|
| Street address |
52 Oxford St. NW 440
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02138 |
| Country |
USA |
| |
|
| Platform ID |
GPL15830 |
| Series (2) |
| GSE39498 |
Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe [ChIP-Seq data] |
| GSE39499 |
Genome-Wide Characterization of the Phosphate Starvation Response in Schizosaccharomyces pombe |
|
| Relations |
| SRA |
SRX160823 |
| BioSample |
SAMN01091695 |
