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| Status |
Public on Aug 10, 2013 |
| Title |
Pou5f1-Flag ChIP 512 cell stage |
| Sample type |
SRA |
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| Source name |
MZspg zebrafish embryos
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| Organism |
Danio rerio |
| Characteristics |
developmental stage: 512 cell stage
tissue: embryo
chip antibody: anti-FLAG® M2
chip antibody vender: Sigma-Aldrich
chip antibody catalog #: F3165
genotype: MZspg injected with Pou5f1-Flag mRNA
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| Growth protocol |
Zebrafish were routinely kept and mated. Eggs were collected and either left untreated (Sox2) or microinjected (Pou5f1; See below). Unfertilized and dead eggs were removed. Eggs were cultivated in 0.3x Danio's at 28.5°C until the appropriate stage was reached.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Embryos were homogenized and fixed with 1% PFA. Cells were lysed and nuclei purified and snap-frozen. 2*10^7 nuclei (Blastula) or 2*10^6 nuclei (preMBT) were pooled and fully lysed. Chromatin was sheared and DNA-Protein complexes were isolated with the respective antibodies and purified. ChIP-library was generated using the Illumina "ChIP-Seq DNA Sample Prep Kit" (Cat. No. IP-102-1001) according to the manufacturers instructions. Nuclei were lysed in 50mM Tris-HCl pH7.5, 10mM EDTA, 1% (w/v) SDS including protease inhibitors (Roche Complete, EDTA free).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls were performed using CASAVA.
Blastula Reads were trimmed to 20bps and aligned to the zv10 genome using "CLC Genomics Workbench" using the following parameters: Match Mode = Random; Sequence Masking = No; Limit = 8; Mismatch Cost = 2; Aligment = Ungapped; Global Aligment = No; Result Handling = Save; pre_MBT reads were similarly aligned but using Mode = Ignore; Using the Input Control Data as background;
Peaks were called using "CLC Genomics Workbench" using the following parameters: Analyze each reference separately = No; Filter peaks on read orientation = No; Filter peaks using Wilcoxon test = No; Shift reads based on fragment length = Yes; Fragment length = 150; Peak refinement = No; Maximum FDR = 5%; Window Size = 50
Peaks were filtered according to following criteria: Normalized read orientation difference <0.5; Wilcoxons test p-value < 0.05. Further, peaks were discarded that showed a larger than 20bp overlap with annotated repeats. Pre_MBT peaks were further filtered more stringently by allowing only peaks with FDR <2.5%
Peaks were normalized to 320 basepairs surrounding the peak center (Average peak size) for applications requiring fixed sequence length.
Genome_build: zv9
Supplementary_files_format_and_content: Filtered peak information was exported from CLC workbench. NOTE: Background peaks (Input Control) are not subtracted yet in these lists. Closest gene TSS in 5' and 3' direction were obtained using Galaxy. Processed data files include the following information: Peak ID, Position (Chromosome, Start, End), Length, False Discovery Rate, Number of total reads, Number of forward reads, Number of reverse reads, Normalized difference between forward and reverse, Wilcoxon p-value, Closest 5' and 3' gene (RefSeq ID)
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| Submission date |
Jun 19, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Manuel Leichsenring |
| Organization name |
Albert-Ludwigs-University Freiburg
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| Street address |
Hauptstr. 1
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| City |
Freiburg |
| ZIP/Postal code |
79104 |
| Country |
Germany |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE39780 |
Zygotic gene activatory network in zebrafish is globally controlled by Pou5f1 SoxB1 enhancers |
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| Relations |
| BioSample |
SAMN02207986 |
| SRA |
SRX310235 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1168898_Peaks_Pou5f1_preMBT.txt.gz |
258.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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