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GEO help: Mouse over screen elements for information. |
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Status |
Public on Jul 20, 2015 |
Title |
InputDNA C2C12-H3mm11-D |
Sample type |
SRA |
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Source name |
C2C12 cells_H3mm11_Differentiated
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Organism |
Mus musculus |
Characteristics |
cell type: myoblast cell line: C2C12 antibody: none (input) culture status: differentiation (D)
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Treatment protocol |
Differentiated cells were transferred to DMEM containing 2% horse serum upon reaching confluence and harvested 48 h later.
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Growth protocol |
Cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 20% fetal bovine serum. Undifferentiated cells were harvested at 60–70% confluence.
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Extracted molecule |
genomic DNA |
Extraction protocol |
Cultured cells were cross-linked in 0.5% formaldehyde and suspended in ChIP buffer (5 mM PIPES, 200 mM KCl, 1 mM CaCl2, 1.5 mM MgCl2, 5% sucrose, 0.5% NP-40, and protease inhibitor cocktail; Nacalai Tesque). Samples were sonicated for 5 s three times, and digested with micrococcal nuclease (1 ul; New England Biolabs, Ipswich, MA) at 37°C for 40 min. The digested samples were centrifuged at 15,000 × g for 10 min. Supernatant containing 4–8 ug DNA was incubated with a rat monoclonal antibody against GFP (1A5, 2 ug, Bio Academia) pre-bound to magnetic beads at 4°C overnight with rotation. The immune complexes were eluted from the beads using 1% SDS in TE, followed by washing with ChIP buffer and TE buffer (both twice). Cross-links were reversed, and DNA was purified using a Qiaquick PCR purification kit (Qiagen, Valencia, CA). ChIP libraries were prepared for sequencing using standard Illumina protocols.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 1500 |
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Description |
Tet-On-C2C12 cells that harbor the N-terminal-GFP-tagged H3mm11 gene under a doxycycline (Dox)-inducible promoter.
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Data processing |
Illumina RTA 1.17 software used for basecalling. Sequenced reads were mapped to the mouse genome (mm9) with STAR alignment software (Dobin et al., 2013) and the parameter “--outFilterMultimapNmax 1 --alignIntronMax 1” (no multi-hit reads, no splice prediction) to treat poly-A containing reads. We defined “ChIP-Seq signal intensity” as described below. First, mapped reads on the genome in a defined window size of 1,000 bp windows by 100 bp intervals; were counted and then normalized as RPKM (reads per kilobases per million reads) (Mortazavi et al., 2008). The ChIP-Seq signal intensities were then calculated as RPKM differences between ChIP and input DNA control data (i.e. ChIP – control) for each bin. genome build: mm9 processed data files format and content: bigWig files of ChIP-Seq signal intensity (as described above) processed data file: w1000s100_H3mm11-D-GFP_sub.bw
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Submission date |
Dec 05, 2014 |
Last update date |
May 15, 2019 |
Contact name |
Yasuyuki Ohkawa |
E-mail(s) |
yohkawa@bioreg.kyushu-u.ac.jp
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Organization name |
Kyushu University
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Department |
Medical Institute of Bioregulation, Medical Institute of Bioregulation
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Street address |
3-1-1 Maidashi, Higashi-ku, Fukuoka
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City |
Fukuoka |
ZIP/Postal code |
812-8582 |
Country |
Japan |
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Platform ID |
GPL18480 |
Series (1) |
GSE63890 |
Characterization of mouse histone H3 variants |
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Relations |
BioSample |
SAMD00019354 |
SRA |
DRX020522 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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