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| Status |
Public on Mar 06, 2017 |
| Title |
WS12h_H3K27ac |
| Sample type |
SRA |
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| Source name |
Cultured limb mesenchymal cells
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| Organism |
Gallus gallus |
| Characteristics |
strain: wild type
cell type: Cultured distal-anterior mesenchymal cells from chick forelimb buds at HH21
hours of incubation: 12
treatment: WNT3A/SHH
antibody: H3K27ac (Abcam ab4729, Lot. GR200563-2)
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| Growth protocol |
Tissues from the distal-anterior portion of 170 forelimb buds were dissected from HH21. After dissection, limb buds were incubated in 0.25% trypsin/PBS for 10 minutes at room temperature to loosen attached ectodermal tissue. Then, tissues were transferred to 10% FBS/DMEM to quench trypsin reaction. Loosened ectodermal tissues were removed and remaining mesenchyme was mechanically dissociated with pipetting. The cell suspension was evenly divided into four tubes and centrifuged at 2,000 rpm for 5 min. After removal of supernatants, the cells were resuspended with 500 μl of DMEM/F12 containing W (250 ng/ml of Wnt3a, R&D system), WF (Wnt3a and 120 ng/ml Fgf8b, R&D system), WS (Wnt3a and 1 ng/ μl of N-terminal fragment of Shh, R&D system), or WFS (Wnt3a, Fgf8b and Shh). 100 μl of cell suspension were plated in each well of 96-well plates (5x10^6 cells/ml), and cultured for 12 hrs at 37°C. After rinsing with PBS, the cultured cells were trypsinized for 5 min at 37°C, and were harvested.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Harvested cells were fixed in 1% formaldehyde/PBS for 15 minutes at room temperature, and the cross-link reaction was quenched by adding Glycine, followed by washing with cold PBS containing proteinase inhibitor. Samples were stored at -80°C before use. The ChIP experiment was performed according to (Beccari et al., 2016) using 3 µg of H3K27ac (ab4729, Abcam).
For ChIP-seq, at least 5 ng of purified DNA were used to make libraries according to the manufacturer’s protocol (Illumina). The material was sequenced with 100 bp single-end reads on the Illumine HiSeq according to the manufacturer’s specifications.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Illumina Casava1.8.2 software used for basecalling. Demultiplexed ChIP-seq reads were mapped onto the modified ICGSC Gallus_gallus-4.0 (galGal4) using Bowtie (Galaxy Tool Version 1.1.2) (Langmead et al., 2009), with parameters “-m1 –strata –best” according to the conditions described previously (Riising et al., 2014).
The coordinates “chr2 --start=32915240 --end=33195168” of galGal4 were masked to take away the second and third copy of the HoxA cluster.
Both unmapped regions containing adaptors and contamination, and PCR duplicates were removed from mapped reads. Three fastq files from W, WF and WFS input data were concatenated and used for further analysis.
These analyses were performed on the public server of the Galaxy Project (https://usegalaxy.org/) (Hillman-Jackson et al., 2012).
By using bamCompare (Galaxy Tool Version 2.4.1.0) from the Galaxy deepTools2 webserver (http://deeptools.ie-freiburg.mpg.de/), ChIP and merged input data were normalized and compared to compute the log2 of the number of reads ratio (Ramirez et al., 2014, 2016).
Genome_build: customized galGal4
Supplementary_files_format_and_content: bedGraph of log2 of the normalized number of reads ratio histone marks ChIP versus concatenated Input.
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| Submission date |
Dec 19, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Nayuta Yakushiji-Kaminatsui |
| E-mail(s) |
nayuta.yakushiji@riken.jp
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| Phone |
+81 45 503 9690
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| Organization name |
RIKEN Center for Integrative Medical Sciences
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| Lab |
Laboratory for Developmental Genetics
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| Street address |
1-7-22 Suehiro-cho, Tsurumi-ku
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| City |
Yokohama |
| State/province |
Kanagawa |
| ZIP/Postal code |
230-0045 |
| Country |
Japan |
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| Platform ID |
GPL19005 |
| Series (2) |
| GSE92555 |
Integration of Shh and Fgf signaling in controlling Hox expression in cultured limb cells [ChIP-seq] |
| GSE92557 |
Integration of Shh and Fgf signaling in controlling Hox expression in cultured limb cells |
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| Relations |
| BioSample |
SAMN06163679 |
| SRA |
SRX2435379 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2432220_WS12h_H3K27ac.bedgraph.gz |
223.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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