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| Status |
Public on Apr 15, 2017 |
| Title |
mock for ScPho4 ChIP with ScPho2 |
| Sample type |
SRA |
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| Source name |
OD600=0.3~0.5 log-phase yeast culture
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: EY2879
type: ChIP (mock)
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| Treatment protocol |
300 mL of OD600=0.3-0.5 cell cultures was cross-linked by adding 8.33 mL of 37% formaldehyde (final formaldehyde concentration = 1%) and incubated with swirling for 10 minutes at room temperature. The sample was then quenched by adding 2.5 M glycine solution to a final concentration of 125 mM, and incubated with swirling for 5 minutes at room temperature. Cells were then collected by centrifugation and washed two times with ice-cold TBS.
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| Growth protocol |
Cells were pre-grown in 10 mM Pi Synthetic Complete (SC) medium to saturation, diluted 1:100 and regrown to saturation. The culture is then diluted to OD600 ~ 0.1 in fresh 10 mM Pi SC medium and grown to OD600 ~ 0.3-0.5 prior to extraction
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The cross-linked cell sample was resuspended in ~1 mL of ChIP lysis buffer (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-Deoxycholate), and mechanically lysed using a mini-Beadbeater-24 (BioSpec) with 0.5 mm Zirconia beads (BioSpec), using the following setting: 12 cycles of 30s bead beating, with 1 min on ice-water bath in between. The lysed sample was fragmented by sonication using Covaris E220 Adaptive Focus system (setting: Duty Factor: 10%; Peak Incident Power: 175; Cycles/Burst: 200; Time:150s). Afterwards, the sample was centrifuged to remove the unsoluable fraction. 5% of the solubalized chromatin was removed at this step to serve as input sample (not subjected to immunoprecipitation, but was reverse cross-linked and subjected to DNA extraction and library preparation). The remaining supernatant containing the solubalized chromatin was subjected to immunoprecipitation. Briefly, the supernatant was incubated with Dynabeads MyOne Streptavidin C1 (Invitrogen) with rotation at 4C overnight. The dynabeads were then washed with each of the following buffers for 2 x 2 minutes: 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). Finally the dynabeads were washed with TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) once for 2 minutes. After washing, cross-linking was reversed by incubation of samples at 65C overnight in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.8% SDS. DNA was then purified using the minElute column (Qiagen).
ChIP-seq library was prepared using the NEB NextUltraII DNA library prep kit, following the manufacturer's protocol.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
LA3
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| Data processing |
Basecalling using bcl2fastq-1.8.3
Sequencing reads were aligned to the corresponding species reference genome using Bowtie (v1.1.1) with the option ‘-m 1 --best –strata’
The resulting SAM files were sortedand indexed using Samtools (v1.2)
Peak calling was performed using GEM v2.6 with the option '--smooth 30 --fold 1.5', using mock samples as the control, and using the default Read Distribution file downloaded from the authors' website.
To generate a visual fold enrichment profile for each ChIP experiment (including the mock samples), we used MACS2's "build signal track" feature to calculate a fold enrichment value at each position of the genome, using the input sample as the control. The resulting "BedGraph" format file was converted to "WIG" format using "bedgraph_to_wig.pl" downloaded from https://gist.github.com/svigneau/8846527. The WIG file was then imported into MochiView v1.6 for visualization.
Genome_build: S. cerevisiae genome version S288C_R64-1-1
Supplementary_files_format_and_content: Peaklist
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| Submission date |
Apr 14, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Bin He |
| E-mail(s) |
emptyhb@gmail.com, bin-he@uiowa.edu
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| Organization name |
the University of Iowa
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| Department |
Biology
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| Lab |
Gene Regulatory Evolution
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| Street address |
129 East Jefferson St., Biology Building 450
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
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| Platform ID |
GPL13821 |
| Series (2) |
| GSE97797 |
Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway [Scer ChIP-seq] |
| GSE97801 |
Evolution of Reduced Co-Activator Dependence Led to Target Expansion of a Starvation Response Pathway |
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| Relations |
| BioSample |
SAMN06756858 |
| SRA |
SRX2737259 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2577541_LA3_mock_ScPho4_PHO2_TTAGGC.wig.gz |
1.1 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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