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| Status |
Public on Jul 05, 2018 |
| Title |
Ino2p_N-lim_rep1 |
| Sample type |
SRA |
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| Source name |
Saccharomyces cerevisiae
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: INO2-TAP constructed from CEN.PK 113-5D by adding a C-terminal CBP-ProtA tag to Ino2p
carbon source: 6% (w/v) glucose
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| Growth protocol |
Cells were cultivated in four different limited media in chemostat: n-lim, glu-lim, et-lim and oxy.glu-lim
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Formaldehyde with a final concentration of 1% (w/v) and distilled water were added to the culture to cross-link protein-DNA complexes with OD600 of 1.0 and a total volume of 100 ml. Cross-linking was performed for 12 min at room temperature with shaking and quenched by adding 2.5 M glycine to a final concentration of 125 mM. After 5 min, cells were washed twice with 20 ml cold TBS (10 mM Tris-HCl, 150 mM NaCl, pH7.5) and frozen with liquid nitrogen. For ChIP-exo, cells were disrupted with glass beads on FastPrep 24 (MP Biomedicals) and the crude cell lysate was sonicated to shear chromatin on Branson digital sonifier 250 (Branson Ultrasonics). After centrifugation, the supernatant containing chromatin fragments was applied to IgG Sepharose 6 Fast Flow beads (GE Healthcare) for immunoprecipitation at 4°C with gentle rocking overnight. NEBNext End Repair Module, NEBNextdA-Tailing Module, NEBNext Quick Ligation Module, PreCR Repair Mix, Lambda exonuclease and RecJf (all from New England Biolabs) were used for on-bead end repair, 3´-dA DNA tailing, first adaptor ligation, nick filling-in and chromatin trimming, respectively. The first adaptors contain unique 6 bp-index sequences. To elute and reverse cross-link the bound complexes, TE buffer containing 1% SDS was added to the beads and samples were incubated overnight at 65°C. After protease K (Thermo Scientific) digestion and DNA extraction with phenol/chloroform/isoamyl alcohol (Amresco, US), the single-strand DNAs were subjected to primer extension using phi29 DNA polymerase (New England Biolabs). The products were dA-tailed and ligated with the second adapter using the same reagents as in on-bead reactions, and then amplified by PCR for 20-22 cycles using Phusion High-Fidelity DNA Polymerase (New England Biolabs). GeneRead Size Selection Kit (QIAGEN) was used to purify DNA before and after the second dA-tailing, second adapter ligation and PCR.
The DNA samples prepared with ChIP-exo contain adaptors and are ready for direct sequencing with Illumina sequencer. The samples were pooled together in equimolar amounts before sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
ChIP-exo: I21
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| Data processing |
ChIP-exo sequencing reads (R1 reads) were mapped to reference genome assembly with Bowtie2 using default settings to generate SAM files.
SAM files were treated with SAMtools option –q 20 to remove low-quality reads and then converted to sorted BAM files.
BAM files were trimmed 70 bp from 3’ end using trimBam (http://genome.sph.umich.edu/wiki/BamUtil:_trimBam) to increase resolution.
GEM was used to identify peaks and compare biological duplicates. The noise level was calculated from averaged noise throughout each replicate.
Bedtools was used to identify the genome wide targets.
Genome_build: R64-2-1 of S. cerevisiae S288C
Supplementary_files_format_and_content: Excel .xlsx file containing summit position, motif, distance to closest gene (ATG) and signal-to-noise values of peaks for all cultivation conditions
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| Submission date |
Oct 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
David Bergenholm |
| E-mail(s) |
david.bergenholm@gmail.com
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| Phone |
703538155
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| Organization name |
Chalmers tekniska högskola
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| Street address |
Axgatan 139
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| City |
Mölndal |
| State/province |
SWEDEN |
| ZIP/Postal code |
43140 |
| Country |
Sweden |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE88941 |
Reconstruction of a Global Transcriptional Regulatory Network for Control of Lipid Metabolism in Yeast by Using Chromatin Immunoprecipitation with Lambda Exonuclease Digestion |
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| Relations |
| BioSample |
SAMN05924924 |
| SRA |
SRX2252363 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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