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| Status |
Public on May 19, 2016 |
| Title |
PFL_E12_WT_H3K27ac_rep1 |
| Sample type |
SRA |
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| Source name |
Forelimb
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| Organism |
Mus musculus |
| Characteristics |
strain: CD1
age: E12.5
tissues: Proximal forelimb
genotype: wild type
antibody: H3K27ac (Abcam ab4729, Lot. GR184558-1)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Micro-dissected proximal and distal segments of E12.5 forelimbs from wild type mice were used for ChIP-seq. Samples were fixed in 1% formaldehyde/PBS for 18 minutes at room temperature, washed at least three times with cold PBS containing protease inhibitor and stored at -80°C. Pools of ten pairs of wild type proximal or distal forelimbs were used for each experiment. The samples were treated with lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl and protease inhibitor) for 10 min on ice, followed by RIPA buffer (TE with 140 mM NaCl, 0.1% deoxycholate, 0.1% SDS, 1% TritonX-100 and protease inhibitor) and fragmented to a range of 200-500 bp by a Biorupter sonication device (Diagenode). PierceTM Protein A/G magnetic beads (88803, Thermo Scientific) were conjugated with each antibody in blocking buffer (PBS, 0.5% Tween20, 0.5% BSA and protease inhibitor) for 4 hrs at 4°C and washed with blocking buffer. Chromatin was incubated with the prepared protein A/G beads overnight at 4°C with rotation. 3 µg of H3K27ac (ab4729, Abcam) and 3 µg of H3K27me3 (07-449, Millipore) were used for each immunoprecipitation. Samples were washed 6 times with RIPA buffer, twice with RIPA/500 mM NaCl buffer, twice with LiCl wash buffer (TE, 250 mM LiCl, 0.5% NP-40 and 0.5% deoxycholate) and twice with TE, followed by eluted in direct elution buffer (10 mM Tris-HCl, 5 mM EDTA, 300 mM NaCl and 0.1% SDS). Cross-links were reversed overnight at 65°C and ChIPed DNA was treated with RNase A and proteinase K, and purified by phenol chloroform extraction.
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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| Description |
Proximal forelimb mouse E12.5 WT_H3K27ac ChIPseq
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| Data processing |
Illumina Casava1.8.2 software used for basecalling.Replicate data set was concatenated together.
Demultiplexed ChIP-seq reads were mapped onto the GRCm38 (mm10) 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). Both unmapped regions containing adaptors and contamination, and PCR duplicates were removed from mapped reads.
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 1.5.9.1.0) from the Galaxy deepTools webserver (http://deeptools.ie-freiburg.mpg.de/), ChIP and input data were normalized and compared to compute the log2 of the number of reads ratio (Ramirez et al., 2014).
Genome_build: mm10
Supplementary_files_format_and_content: bedGraph of log2 of the normalized number of reads ratio histone marks ChIP versus Input.
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| Submission date |
Feb 13, 2016 |
| Last update date |
Nov 12, 2020 |
| 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 |
GPL17021 |
| Series (2) |
| GSE77900 |
A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus |
| GSE79261 |
A role for HOX13 proteins in the regulatory switch between TADs at the HoxD locus |
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| Relations |
| Reanalyzed by |
GSE161377 |
| SRA |
SRX1584368 |
| BioSample |
SAMN04501732 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2061120_WT_FLProx_concatenate_H3K27ac.bedgraph.gz |
482.0 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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