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| Status |
Public on Oct 18, 2013 |
| Title |
Pol II ChIP-seq dome 8WG16 |
| Sample type |
SRA |
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| Source name |
embryos
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| Organism |
Danio rerio |
| Characteristics |
tissue: embryos
developmental stage: 4.5 hpf (dome/30% epiboly stage)
chip antibody lot number: E12CF00481
chip antibody: Pol II (8WG16/Covance MMS-126R)
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| Growth protocol |
Zebrafish were maintained and raised under standard conditions. Wild-type embryos were collected at the 1-cell stage, synchronized and allowed to develop to the desired stage at 28°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For Pol II ChIP-seq samples, between 400 and 800 embryos were carefully staged, dechorionated and fixed in 1.85% formaldehyde and 12.5% DMSO for 15 minutes at room temperature. Formaldehyde was quenched by adding glycine to a final concentration of 0.125M. Embryos were rinsed 3 times in ice-cold PBS, immediately resuspended in cell lysis buffer (10mM Tris-HCl pH7.5/10mM NaCl/0.5%NP40) and lysed for 15 min on ice. Nuclei were collected by centrifugation, resuspended in nuclei lysis buffer (50mM Tris-HCl pH7.5/10mM EDTA/1%SDS) and lysed for 10 min on ice. Samples were diluted 3 times in IP dilution buffer (16.7mM Tris-HCl pH7.5/167mM NaCl/1.2mM EDTA/0.01%SDS) and sonicated to obtain fragments of ~400 bp. Triton X-100 was added to a final concentration of 0.75% and the lysate was incubated overnight while rotating at 4°C with 25μl of protein G magnetic Dynabeads (Invitrogen) that had been pre-bound to an excess amount of antibody. Bound complexes were extensively washed with RIPA (50mM HEPES pH7.6/1mM EDTA/0.7%DOC/1%Igepal/0.5MLiCl) and TBS and then eluted from the beads with elution buffer (50mM NaHCO3/1%SDS). Cross-links were reversed o/n at 65°C, and DNA purified by QIAquick PCR purification kit (QIAGEN). For MNase/ChIP between 300 and 700 embryos were used per condition and after dialysis, chromatin was added to the bead/antibody complex as described above.
Libraries were prepared using the Illumina sequencing library preparation protocol.
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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 |
All sequenced reads were aligned using bowtie version 4.1.2.
Uniquely mapped reads with a maximum of two mismatches were kept.
For nucleosome and histone modification ChIP-seq samples, all mapped reads were extended to 147 bp in their 3’ direction, and the middle 73 bp were piled up to generate wig files.
For Pol II ChIP-seq samples, MACS version 1.4.2 was used to generate wig files.
Genome_build: zv9
Supplementary_files_format_and_content: wig format
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| Submission date |
Feb 12, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Yong Zhang |
| Organization name |
Tongji University
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| Street address |
1239 Siping Road
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| City |
Shanghai |
| ZIP/Postal code |
200092 |
| Country |
China |
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| Platform ID |
GPL14875 |
| Series (1) |
| GSE44269 |
Canonical Nucleosome Organization at Promoters Forms During Genome Activation |
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| Relations |
| SRA |
SRX235873 |
| BioSample |
SAMN01919767 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1081560_Pol_II_ChIP-seq_dome_8WG16.wig.gz |
244.0 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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