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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 06, 2008 |
| Title |
abMyc |
| Sample type |
genomic |
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| Channel 1 |
| Source name |
antibody Myc ChIP DNA from ES cells (3 repeats)
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| Organism |
Mus musculus |
| Characteristics |
J1 ES cells
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| Growth protocol |
Normal J1 ES growth condition
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For biotin-mediated ChIP, approximately, 5 × 107 mES cells expressing both BirA and biotinylated proteins were used. Briefly, cells were cross-linked by addition of final 1% formaldehyde for 7 min at room temperature. Cross-linking was terminated by adding final 125 mM glycine and cells were washed with cold phosphate-buffered saline (PBS) containing PMSF, scraped off the plates, collected by centrifugation and washed again. Collected cell pellet was resuspended in SDS ChIP buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl pH 8.1, 150 mM NaCl, and protease inhibitors). Cells were sonicated and fragmented DNA was visualized on an agarose gel (average size 0.5-1 kb). The sample was centrifuged at 12000 rpm at 4°C for 10 min and supernatant was collected. Sample was pre-cleared with protein A beads at 4°C for 1 hr and incubated with streptavidin beads (Dynabeads® MyOne™ Streptavidin T1) at 4°C overnight. For reference sample, J1 ES cells expressing BirA enzyme without biotinylated protein were used. Immunoprecipitated complexes were successively washed with buffer I (2% SDS), buffer II (0.1% Deoxycholate, 1% Triton X-100, 1 mM EDTA, 50 mM HEPES pH 7.5, 500 mM NaCl), buffer III (250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA, 10 mM Tris-Cl pH 8.1) and TE buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA). All washes were for 8 min at room temperature. SDS elution buffer was added and incubated at 65°C overnight to reverse crosslink protein-DNA complexes. The sample was treated with RNase A and Proteinase K, extracted with phenol:chloroform and precipitated. The pellet was resuspended in 25μl of water.
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| Label |
biotin
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| Label protocol |
Each ChIP was amplified using ligation-mediated PCR (LM-PCR). Labeling was according to standard Affymetrix protocols.
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| Channel 2 |
| Source name |
Input DNA from normal J1 ES cells (3 repeats)
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| Organism |
Mus musculus |
| Characteristics |
Reference for antibodyChIP
J1 ES cells
|
| Growth protocol |
Normal J1 ES growth condition
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For biotin-mediated ChIP, approximately, 5 × 107 mES cells expressing both BirA and biotinylated proteins were used. Briefly, cells were cross-linked by addition of final 1% formaldehyde for 7 min at room temperature. Cross-linking was terminated by adding final 125 mM glycine and cells were washed with cold phosphate-buffered saline (PBS) containing PMSF, scraped off the plates, collected by centrifugation and washed again. Collected cell pellet was resuspended in SDS ChIP buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl pH 8.1, 150 mM NaCl, and protease inhibitors). Cells were sonicated and fragmented DNA was visualized on an agarose gel (average size 0.5-1 kb). The sample was centrifuged at 12000 rpm at 4°C for 10 min and supernatant was collected. Sample was pre-cleared with protein A beads at 4°C for 1 hr and incubated with streptavidin beads (Dynabeads® MyOne™ Streptavidin T1) at 4°C overnight. For reference sample, J1 ES cells expressing BirA enzyme without biotinylated protein were used. Immunoprecipitated complexes were successively washed with buffer I (2% SDS), buffer II (0.1% Deoxycholate, 1% Triton X-100, 1 mM EDTA, 50 mM HEPES pH 7.5, 500 mM NaCl), buffer III (250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA, 10 mM Tris-Cl pH 8.1) and TE buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA). All washes were for 8 min at room temperature. SDS elution buffer was added and incubated at 65°C overnight to reverse crosslink protein-DNA complexes. The sample was treated with RNase A and Proteinase K, extracted with phenol:chloroform and precipitated. The pellet was resuspended in 25μl of water.
|
| Label |
biotin
|
| Label protocol |
Each ChIP was amplified using ligation-mediated PCR (LM-PCR). Labeling was according to standard Affymetrix protocols.
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| Hybridization protocol |
According to standard Affymetrix protocols.
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| Scan protocol |
According to standard Affymetrix protocols.
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| Description |
antibodyChIP
abMyc_1.CEL
abMyc_2.CEL
abMyc_3.CEL
Input_J1_1.CEL
Input_J1_2.CEL
Input_J1_3.CEL
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| Data processing |
MAT (Johnson et al., 2006) was used to process the raw CEL files. The following settings; Bandwith = 300, MaxGap = 300, MinProbe = 10 were used. Initially BAR and BED files were generated by using Mm_PromPR_v02-1_NCBIv33.bpmap and genomic coordinates in BED files were converted to new coordinates based on mouse genome assembly mm8.
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| Submission date |
May 02, 2008 |
| Last update date |
May 11, 2018 |
| Contact name |
Jonghwan Kim |
| E-mail(s) |
jkim@bloodgroup.tch.harvard.edu, jybella@yahoo.com
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| Phone |
607-919-2051
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| Organization name |
HMS/CHB
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| Lab |
Stuart H Orkin lab
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| Street address |
1 Blackfan circle Karp-7
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL5811 |
| Series (1) |
| GSE11329 |
Chip-chip from mouse embryonic stem (mES) cells |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM286128_Input_J1_1.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286128_Input_J1_2.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286128_Input_J1_3.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286128_abMyc.bar |
31.7 Mb |
(ftp)(http) |
BAR |
| GSM286128_abMyc.bed |
333.6 Kb |
(ftp)(http) |
BED |
| GSM286128_abMyc_1.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286128_abMyc_2.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286128_abMyc_3.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| Processed data provided as supplementary file |
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