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| Status |
Public on Dec 02, 2013 |
| Title |
ChIP-seq H3K4me3 in bud-stage embryo |
| Sample type |
SRA |
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| Source name |
Bud stage embryo
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| Organism |
Danio rerio |
| Characteristics |
tissue: whole embryo
strain: Tu
chip antibody: Anti-Trimethyl-Histone H3 (Lys4) (Millipore 07-473)
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| Treatment protocol |
Wild-type (Tu) embryos were collected at the 1-cell stage, and were kept uninjected, or injected with mRNA for further experiments.
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| Growth protocol |
Zebrafish were maintained and raised under standard conditions.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each ChIP experiment, ~2000 embryos were carefully staged. 500 embryos were then collected, transferred into PBS containing 20mM Na-butyrate, protease inhibitors (Sigma). Embryos were then transferred to 5ml syringe fitted with 21G needle. After leaving ~500ul buffer above the embryos, embryos were forced through the needle by pushing the pistons to dissociate the embryos. The resulting lysed extract was collected in 2ml eppendorf tubes, and crosslinked by adding formaldehyde solution to final concentration of 1.1%. Crosslinking was conducted for 15 minutes at room temperature while samples were mixed using nutator. After crosslinking, glycine was added to 0.125M to quench the formaldehyde, and incubated on ice for 5 minutes. Tubes were then centrifuged at 470g for 10 minutes at 4C to sediment the cells. The cells were then washed twice with 500ul ice-cold PBS/Na-butyrate/protease inhibitor solution, and flash-frozen and kept at -80C until the ChIP experiment.
ChIP-seq libraries prepared using standard Illumina protocols
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Chromatin IP against H3K4me3
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| Data processing |
Basecalls performed using CASAVA version 1.4
Alignment: sequencing reads were aligned to the zebrafish genome (version danRer6) using Bowtie version 0.12.7 with options "-q --best --strata -m 1 -p 4 --chunkmbs 1024", and only uniquely mapping reads were retained for further analysis.
Peak-calling: Binding events were detected using GPS as previously described (Guo et al. 2010). In GPS, the scaling ratio between IP and control channels was estimated using the median ratio of all 10Kbp windows along the genome. The GPS binding model was initialized to the default and iteratively updated over up to 3 training rounds, and the shape deviation threshold for final peaks was set to -0.2. In this study, we require that reported peaks contain a ChIP-seq enrichment level that is significantly greater than 1.5 times the control channel read count over the same region with p-value <0.001 as tested using a Binomial test.
Domain-calling: Histone mark domains were detected using a 200bp sliding window (offset = 100bp) along the genome, merging neighboring windows that individually contain significantly more signal reads than expected by a global Poisson background model (p<10^-9) and significantly more signal reads than expected by local Poisson background models parameterized by the input samples (p<10^-9).
Genome_build: danRer6
Supplementary_files_format_and_content: BED: Cdx4 and Sall4 peaks.bed files are conversions of GPS output files to BED format, centered on the GPS binding event location and extended to 100bp width. Chromatin enriched domains are also output in BED format.
Supplementary_files_format_and_content: BIGWIG: WIG files were generated using custom software to summarize read counts overlapping genomic regions. All reads were artificially extended out to 200bp in length. A bin size of 20bp was used for calculating overlapping read counts. WIG files were converted to bigWig using wigToBigWig (UCSC)
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| Submission date |
Jun 24, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Shaun Mahony |
| E-mail(s) |
mahony@psu.edu
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| Phone |
814-865-3008
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| Organization name |
Penn State University
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| Department |
Biochemistry & Molecular Biology
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| Lab |
Shaun Mahony
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| Street address |
404 South Frear Bldg
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| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE48254 |
A Cdx4-Sall4 regulatory module controls the transition from mesoderm formation to embryonic hematopoiesis |
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| Relations |
| BioSample |
SAMN02212609 |
| SRA |
SRX314761 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1173382_Zon_Bud_H3K4me3.domains.bed.gz |
204.8 Kb |
(ftp)(http) |
BED |
| GSM1173382_Zon_Bud_H3K4me3_120103_s4-1.bw |
58.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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