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| Status |
Public on Dec 31, 2014 |
| Title |
Mo-DC H4Ac LPS 1 hour rep 2.1 |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
H4Ac ChIP DNA from immature DC
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| Organism |
Homo sapiens |
| Characteristics |
cell type: Monocyte-derived denditric cells
chip antibody: H4Ac
|
| Treatment protocol |
Untreated Monocyte-derived dendritic cells
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| Growth protocol |
monocytes were cultured in RPMI 10% FCS supplemented with 2-mercaptoethanol (50 μM), GM-CSF (600 U/ml), and IL-4 (750 U/ml)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were fixed with 1% formaldehyde during 8 minutes followed by quenching with 0.18 M glycine.To isolate chromatin, the cell pellet was resuspended in 20 ml of TE supplemented with 0.5% NP-40, 1mM PMSF, 5mM benzamidine, 1ug/ml leupeptin, and 5ug/ml aprotinin, centrifuged as above, resuspended in 20 ml of TE supplemented with 1% Triton X-100, 0.5% Na-DOC, 0.5M NaCl, 1mM PMSF, 5mM benzamidine, 1ug/ml leupeptin, and 5ug/ml aprotinin. To break up the chromatin into ~300 to ~600bp fragments, the pellet was resuspended in 1.5ml of TEN (TE with 0.1M NaCl) and sonicated on ice. For one immunoprecipitation reaction, 10 ug of chromatin was diluted into 300ul of dilution/incubation buffer (20mM HEPES pH 7.9, 0.2M NaCl, 2mM EDTA, 0.1% Na-DOC, 0.1% SDS,1mg/ml BSA, Complete™ cocktail of protease inhibitors) and pre-cleared by the addition of 12ul of a 50:50 slurry of protein A Sepharose CL-4B beads (pre-washed in dilution/incubation buffer) and a 30 minute incubation with tumbling at room temperature. The supernatant was cleared by centrifugation and brought up to 1ml with dilution/incubation buffer. Antibodies were added and the reaction was incubated overnight with slow tumbling at 4 degress. The next day, the reactions were cleared by a 10 minute centrifugation at 10krpm at room temperature, 12 ul of a 50:50 slurry of protein A Sepharose CL-4B beads (pre-washed as before) was added and the reaction was incubated for 1h at room temperature with tumbling. The immunoprecipitates were washed twice in 20mM HEPES 7.9, 0.2M NaCl, 2mM EDTA, 0.1% Na-DOC, 0.1% SDS, twice in 20mM HEPES 7.9, 0.5M NaCl, 2mM EDTA, 0.1% Na-DOC, 0.1% SDS, twice in 20mM Tris-HCl 8.0, 0.25M LiCl, 2mM EDTA, 0.5% Na-DOC, 0.5% NP-40 and once in TE-0.1% NP-40. To reverse cross-links, 500ul of elution buffer (110mM Tris 8.0, 1.1% SDS) was added to the reaction and to a 10ug aliquot of non-immunoprecipitated chromatin (input). The sample was incubated for 10 minutes at 65 degress, 14ul of proteinase K (10mg/ml) and 11ul of 5M NaCl were added, the sample was cleared by centrifugation and then incubated at 42 degress for 2h and at 65 degress overnight. To extract DNA, one phenol-chloroform and one chloroform extraction were done. DNA was then precipitated by 20ug glycogen, 66ul NaAc (3M, pH 5.2) and 900 ul of isopropanol and resuspended in 100 ul of TE.
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| Label |
Cy5
|
| Label protocol |
manufacturer's protocol
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| Channel 2 |
| Source name |
H4Ac ChIP DNA from 1h LPS DC
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Monocyte-derived denditric cells
chip antibody: H4Ac
|
| Treatment protocol |
1 hour LPS treated Monocyte-derived dendritic cells
|
| Growth protocol |
monocytes were cultured in RPMI 10% FCS supplemented with 2-mercaptoethanol (50 μM), GM-CSF (600 U/ml), and IL-4 (750 U/ml)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were fixed with 1% formaldehyde during 8 minutes followed by quenching with 0.18 M glycine.To isolate chromatin, the cell pellet was resuspended in 20 ml of TE supplemented with 0.5% NP-40, 1mM PMSF, 5mM benzamidine, 1ug/ml leupeptin, and 5ug/ml aprotinin, centrifuged as above, resuspended in 20 ml of TE supplemented with 1% Triton X-100, 0.5% Na-DOC, 0.5M NaCl, 1mM PMSF, 5mM benzamidine, 1ug/ml leupeptin, and 5ug/ml aprotinin. To break up the chromatin into ~300 to ~600bp fragments, the pellet was resuspended in 1.5ml of TEN (TE with 0.1M NaCl) and sonicated on ice. For one immunoprecipitation reaction, 10 ug of chromatin was diluted into 300ul of dilution/incubation buffer (20mM HEPES pH 7.9, 0.2M NaCl, 2mM EDTA, 0.1% Na-DOC, 0.1% SDS,1mg/ml BSA, Complete™ cocktail of protease inhibitors) and pre-cleared by the addition of 12ul of a 50:50 slurry of protein A Sepharose CL-4B beads (pre-washed in dilution/incubation buffer) and a 30 minute incubation with tumbling at room temperature. The supernatant was cleared by centrifugation and brought up to 1ml with dilution/incubation buffer. Antibodies were added and the reaction was incubated overnight with slow tumbling at 4 degress. The next day, the reactions were cleared by a 10 minute centrifugation at 10krpm at room temperature, 12 ul of a 50:50 slurry of protein A Sepharose CL-4B beads (pre-washed as before) was added and the reaction was incubated for 1h at room temperature with tumbling. The immunoprecipitates were washed twice in 20mM HEPES 7.9, 0.2M NaCl, 2mM EDTA, 0.1% Na-DOC, 0.1% SDS, twice in 20mM HEPES 7.9, 0.5M NaCl, 2mM EDTA, 0.1% Na-DOC, 0.1% SDS, twice in 20mM Tris-HCl 8.0, 0.25M LiCl, 2mM EDTA, 0.5% Na-DOC, 0.5% NP-40 and once in TE-0.1% NP-40. To reverse cross-links, 500ul of elution buffer (110mM Tris 8.0, 1.1% SDS) was added to the reaction and to a 10ug aliquot of non-immunoprecipitated chromatin (input). The sample was incubated for 10 minutes at 65 degress, 14ul of proteinase K (10mg/ml) and 11ul of 5M NaCl were added, the sample was cleared by centrifugation and then incubated at 42 degress for 2h and at 65 degress overnight. To extract DNA, one phenol-chloroform and one chloroform extraction were done. DNA was then precipitated by 20ug glycogen, 66ul NaAc (3M, pH 5.2) and 900 ul of isopropanol and resuspended in 100 ul of TE.
|
| Label |
Cy3
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| Label protocol |
manufacturer's protocol
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| |
| Hybridization protocol |
manufacturer's protocol
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| Scan protocol |
manufacturer's protocol
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| Description |
comparison of DCi/ DCm H4ac after 1 hour LPS treatment
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| Data processing |
manufacturer's data processing
normalized log2 ratios (Cy5/Cy3)
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| Submission date |
Jul 01, 2014 |
| Last update date |
Dec 31, 2014 |
| Contact name |
Mark Ibberson |
| Organization name |
SIB Swiss Institute of Bioinformatics
|
| Department |
Vital-IT
|
| Street address |
Genopode building
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| City |
Lausanne |
| ZIP/Postal code |
CH-1015 |
| Country |
Switzerland |
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| Platform ID |
GPL7408 |
| Series (2) |
| GSE58961 |
Extensive remodeling of DC function by rapid maturation-induced epigenetic gene silencing [ChIP-chip] |
| GSE59365 |
Extensive remodeling of DC function by rapid maturation-induced epigenetic gene silencing |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM1423104_Exp2_DCi_cy5_p1.pair.gz |
6.6 Mb |
(ftp)(http) |
PAIR |
| GSM1423104_Exp2_DCm_cy3_p1.pair.gz |
6.6 Mb |
(ftp)(http) |
PAIR |
| GSM1423104_Exp2_p1_ratio.gff.gz |
4.8 Mb |
(ftp)(http) |
GFF |
| Processed data provided as supplementary file |
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