|
| Status |
Public on Jul 05, 2012 |
| Title |
MyoD-10h |
| Sample type |
SRA |
| |
|
| Source name |
myocytes with TAP tagged MyoD, 10h in differentiating medium
|
| Organism |
Mus musculus |
| Characteristics |
strain: Balb/c
genotype/variation: wild type
tissue of origin: skeletal muscle
cell type: myocytes
transcription factor: MyoD
growth protocol: 10h in differentiating medium
sample type: ChIP
flag immunoprecipitation: anti-FLAG M2 agarose resin
6xhis immunoprecipitation: His-Select nickel beads
|
| 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 |
Pulldown of FLAG and TAP tagged MyoD. 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 |
| SRA |
SRX029145 |
| BioSample |
SAMN00116436 |
