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Sample GSM611281 Query DataSets for GSM611281
Status Public on Jul 05, 2012
Title EV-GM
Sample type SRA
 
Source name cycling myoblasts with empty vector
Organism Mus musculus
Characteristics strain: Balb/c
genotype/variation: wild type
tissue of origin: skeletal muscle
cell type: myoblasts
transcription factor: control (empty vector)
sample type: control
Growth protocol HAMS's F10 + 20% FBS + bFGF(4ng/ml)
Extracted molecule genomic DNA
Extraction protocol Cycling cells or differentiated myotubes stably expressing a c-terminus TAP tagged fusion proteins were cross linked with with 1% formaldehyde in 1x PBS for 10 minutes. A solution containing 0.125 M glycine in 1x PBS was used for quenching for 5 minutes at room temperature. Cells were harvested and the pellet dissolved in ChIP lysis buffer (40 mM Tris-HCl, pH8.0; 1% Triton-X100; 4 mM EDTA; 300 mM NaCl) containing protease inhibitors. Chromatin was fragmented by sonication in a water bath sonicator at 4 ºC to an average length of 200 base pairs. The lysate was spun at 14K for 15 minutes and the supernatant was diluted 1:1 in ChIP dilution buffer containing 40 mM Tris-HCl, pH 8.0; 4 mM EDTA plus protease inhibitors. FLAG immunoprecipitation was done on 15 milligram of cell lysate using M2 conjugated agarose beads (Sigma Aldrich) for 2 hours at 4 ºC following manufacture’s recommendation. Beads were washed 3 times with 10 mM Tris-HCl, pH 8.0; 100 mM NaCl; 0.1% Triton-X100 plus protease inhibitors. Protein complex was eluted from M2-conjugated agarose beads using proteolytic cleavage with Tobacco Etch Virus (TEV) protease (Invitrogen) together with 3xFLAG peptide (Sigma Aldrich) competition over night at 4 ºC using 1x TEV buffer (Invitrogen). Two additional rounds of elution with 3xFLAG peptides were done using 200 µg/ml of 3xFLAG peptide in TBS (50 mM Tris-HCl, pH 7.4; 150 mM NaCl). 6xHis immunoprecipitation and purification was done using His-Select nickel beads (Sigma-Aldrich) for three hours at 4 ºC following manufacture’s recommendations. Beads were washed three times with wash buffer (20 mM Tris-HCl, pH 7.4; 150 mM NaCl; 5 mM Imidazole plus protease inhibitors). Final DNA/protein complex was eluted by 400 mM imidazole at room temperature. Reverse cross linking and phenol/chloroform extraction of chromatin were performed. The ChIP DNA library was prepared according to the Illumina protocol (www.illumina.com)
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina Genome Analyzer II
 
Description Control run with no tagged proteins. Instrument model: Illumina GA-IIx
Data processing Solexa read alignment and peak calling. Solexa reads (36 bp) were aligned to mouse genome (NCBI37) by Eland (Illumina) with up to two mismatches.
 
Submission date Oct 21, 2010
Last update date May 15, 2019
Organization Ottawa Hospital Research Institute
Phone (613) 737-8899 -73255
Department Cellular and Molecular Medicine
Lab Ottawa Bioinformatics Core Facility
Street address 501 Smyth Rd.
City Ottawa
State/province ON
ZIP/Postal code K1H 8L6
Country Canada
 
Platform ID GPL9250
Series (2)
GSE24852 ChIP-Seq of Myf5, MyoD, Snai1, HDAC1, HDAC2, E47 and empty vector controls in mouse skeletal myoblasts or myotubes
GSE24904 Snail regulates MyoD binding-site occupancy to direct enhancer switching and differentiation-specific transcription in myogenesis
Relations
Reanalyzed by GSE80791
SRA SRX029148
BioSample SAMN00116439

Supplementary file Size Download File type/resource
GSM611281_84_s_3_export.txt.gz 954.7 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data included within Sample table

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