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Sample GSM1559511 Query DataSets for GSM1559511
Status Public on Jul 20, 2015
Title ChIP GFP C2C12-H3.3-D
Sample type SRA
 
Source name C2C12 cells_H3.3_Differentiated
Organism Mus musculus
Characteristics cell type: myoblast
cell line: C2C12
antibody: rat monoclonal antibody against GFP (1A5, 2 ug, Bio Academia)
culture status: differentiation (D)
Treatment protocol Differentiated cells were transferred to DMEM containing 2% horse serum upon reaching confluence and harvested 48 h later.
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.
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.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 1500
 
Description Tet-On-C2C12 cells that harbor the N-terminal-GFP-tagged H3.3 gene under a doxycycline (Dox)-inducible promoter.
Data processing Illumina RTA 1.17 software used for basecalling.
The sequence reads for GFP, and Input were aligned to the reference mouse genome (mm9, build 37) using Bowtie 2 software (version 2.2.2) (Langmead and Salzberg, 2012). PCR duplicates were removed from uniquely mapped reads using samtools (version 0.1.19).
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_H3v3-D-GFP_sub.bw
 
Submission date Dec 05, 2014
Last update date May 15, 2019
Contact name Yasuyuki Ohkawa
E-mail(s) yohkawa@bioreg.kyushu-u.ac.jp
Organization name Kyushu University
Department Medical Institute of Bioregulation, Medical Institute of Bioregulation
Street address 3-1-1 Maidashi, Higashi-ku, Fukuoka
City Fukuoka
ZIP/Postal code 812-8582
Country Japan
 
Platform ID GPL18480
Series (1)
GSE63890 Characterization of mouse histone H3 variants
Relations
BioSample SAMD00019353
SRA DRX020502

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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