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| Status |
Public on Jul 05, 2012 |
| Title |
GA184.7_Hdac2_GM |
| Sample type |
SRA |
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| Source name |
TAP-tagged HDAC2 in Myoblasts
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| Organism |
Mus musculus |
| Characteristics |
cell type: Myoblasts
strain: C57BL/6
transcription factor: HDAC2
tissue: skeletal muscle
flag immunoprecipitation: anti-FLAG M2 agarose resin
6xhis immunoprecipitation: His-Select nickel beads
sample type: ChIP
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| Treatment protocol |
For each transcription factor cTAP were engineered with the in-frame addition of a C-terminal TAP tag, composed of 6 histidine residues and a 3x FLAG tag separated by a Tobacco Etch Virus (TEV) cleavage sequence. Retrovirus was produced in 293FT cells by transient transfection of expression constructs using Calcium Phosphate or linear polyethylenimine (PEI). Virus-containing supernatant was used to infect low-passage primary myoblasts supplemented with Ham’s complete medium and 8µg/ml polybrene. Transgenic cells were obtained by antibiotic selection and maintained in Ham’s complete medium containing 1.0 µg/ml puromycin. Satellite cell-derived myoblasts stably expressing a C-terminus TAP tagged fusion protein were cross-linked using 1% formaldehyde in 1x PBS for 10 minutes. A solution containing 0.125 M glycine in 1x PBS was used for quenching the cross-linked reaction for 5 minutes at room temperature by gentle horizontal shaking. Cells were harvested by scraping and the cell pellet was dissolved in ChIP lysis buffer (40 mM Tris-HCl, pH 8.0; 1% Triton-X100; 4 mM EDTA; 300 mM NaCl) containing protease inhibitors (Aprotinin, 1 µg/ml; Leupeptin, 5µg/ml, Pepstatin A 1 µg/ml; PMSF, 1mM). Chromatin was fragmented by sonication in a water bath Bioruptor at 4ºC to an average length of 200 base pairs. The lysate was spun at 14K RPM for 15 minutes in a refrigerated centrifuge and the supernatant was diluted 1:1 in ChIP dilution buffer (40 mM Tris-HCl, pH 8.0; 4 mM EDTA, pH 8.0) plus protease inhibitors to reduce the concentration of salt and detergents. The first immunoprecipitation was done using anti 3xFLAG antibody conjugated to agarose beads (Sigma Aldrich) using 20 milligram of cell lysate (protein) as input for 2 hours at 4ºC. Beads containing antigen/antibody complex were washed 3 times with 10 mM Tris-HCl, pH 8.0; 100 mM NaCl; 0.1% Triton-X100 containing protease inhibitors. Protein complex was eluted from M2-conjugated agarose beads using proteolytic cleavage with Tobacco Etch Virus (TEV) protease (Invitrogen) together with competition with 3xFLAG peptide (Sigma Aldrich) at 4ºC overnight. 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). The eluted product was used as input for the second affinity pull-down using His-Select nickel beads (Sigma-Aldrich) for three hours at 4ºC 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). Final DNA/protein complex was eluted by 400 mM imidazole at room temperature. DNA/protein complexes were reverse cross-linked and ChIP DNA was purified by phenol/chloroform extraction followed by ethanol precipitation.
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| Growth protocol |
Muscle satellite cells were isolated from adult C57BL/6 mice and cultured on collagen-coated plates (Roche-Boehringer) in Ham’s complete medium (Ham’s F-10, 20% fetal calf serum, 1% penicillin/streptomycin, and 2.5ng/ml human recombinant bFGF.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each transcription factor cTAP were engineered with the in-frame addition of a C-terminal TAP tag, composed of 6 histidine residues and a 3x FLAG tag separated by a Tobacco Etch Virus (TEV) cleavage sequence. Retrovirus was produced in 293FT cells by transient transfection of expression constructs using Calcium Phosphate or linear polyethylenimine (PEI). Virus-containing supernatant was used to infect low-passage primary myoblasts supplemented with Ham’s complete medium and 8µg/ml polybrene. Transgenic cells were obtained by antibiotic selection and maintained in Ham’s complete medium containing 1.0 µg/ml puromycin. Satellite cell-derived myoblasts stably expressing a C-terminus TAP tagged fusion protein were cross-linked using 1% formaldehyde in 1x PBS for 10 minutes. A solution containing 0.125 M glycine in 1x PBS was used for quenching the cross-linked reaction for 5 minutes at room temperature by gentle horizontal shaking. Cells were harvested by scraping and the cell pellet was dissolved in ChIP lysis buffer (40 mM Tris-HCl, pH 8.0; 1% Triton-X100; 4 mM EDTA; 300 mM NaCl) containing protease inhibitors (Aprotinin, 1 µg/ml; Leupeptin, 5µg/ml, Pepstatin A 1 µg/ml; PMSF, 1mM). Chromatin was fragmented by sonication in a water bath Bioruptor at 4ºC to an average length of 200 base pairs. The lysate was spun at 14K RPM for 15 minutes in a refrigerated centrifuge and the supernatant was diluted 1:1 in ChIP dilution buffer (40 mM Tris-HCl, pH 8.0; 4 mM EDTA, pH 8.0) plus protease inhibitors to reduce the concentration of salt and detergents. The first immunoprecipitation was done using anti 3xFLAG antibody conjugated to agarose beads (Sigma Aldrich) using 20 milligram of cell lysate (protein) as input for 2 hours at 4ºC. Beads containing antigen/antibody complex were washed 3 times with 10 mM Tris-HCl, pH 8.0; 100 mM NaCl; 0.1% Triton-X100 containing protease inhibitors. Protein complex was eluted from M2-conjugated agarose beads using proteolytic cleavage with Tobacco Etch Virus (TEV) protease (Invitrogen) together with competition with 3xFLAG peptide (Sigma Aldrich) at 4ºC overnight. 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). The eluted product was used as input for the second affinity pull-down using His-Select nickel beads (Sigma-Aldrich) for three hours at 4ºC 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). Final DNA/protein complex was eluted by 400 mM imidazole at room temperature. DNA/protein complexes were reverse cross-linked and ChIP DNA was purified by phenol/chloroform extraction followed by ethanol precipitation.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Data processing |
GERALD within CASAVA version 1.6.0 (GA184.8_E47_2d,GA184.7_Hdac2_GM,SC0017.2_Snai1_GM,SC0017.4_EV_GM)
GERALD within CASAVA version 1.7.0 (HI022.3_Hdac1_GM)
Genome_build: mm9
Supplementary_files_format_and_content: Illumina ELAND format
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| Submission date |
May 25, 2012 |
| Last update date |
May 15, 2019 |
| Organization |
Ottawa Hospital Research Institute |
| Phone |
(613) 737-8899 -73255
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| Department |
Cellular and Molecular Medicine
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| Lab |
Ottawa Bioinformatics Core Facility
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| Street address |
501 Smyth Rd.
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| City |
Ottawa |
| State/province |
ON |
| ZIP/Postal code |
K1H 8L6 |
| Country |
Canada |
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| Platform ID |
GPL11002 |
| 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 |
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| Relations |
| SRA |
SRX150193 |
| BioSample |
SAMN00998877 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM937913_HDAC2_GM.bw |
3.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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