 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 06, 2008 |
| Title |
bioNanog |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
bioNanog ChIP DNA from ES cells (3 repeats)
|
| Organism |
Mus musculus |
| Characteristics |
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.
|
| |
|
| Channel 2 |
| Source name |
Mock biotin ChIP DNA from ES cells expression BirA (4 repeats)
|
| Organism |
Mus musculus |
| Characteristics |
Reference for bioChIP
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.
|
| |
|
| |
| Hybridization protocol |
According to standard Affymetrix protocols.
|
| Scan protocol |
According to standard Affymetrix protocols.
|
| Description |
bioChIP
bNanog_1.CEL
bNanog_2.CEL
bNanog_3.CEL
BirA_1.CEL
BirA_2.CEL
BirA_3.CEL
BirA_4.CEL
|
| 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.
|
| |
|
| 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
|
| Phone |
607-919-2051
|
| Organization name |
HMS/CHB
|
| Lab |
Stuart H Orkin lab
|
| Street address |
1 Blackfan circle Karp-7
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
| |
|
| Platform ID |
GPL5811 |
| Series (1) |
| GSE11329 |
Chip-chip from mouse embryonic stem (mES) cells |
|
| Supplementary file |
Size |
Download |
File type/resource |
| GSM286118_BirA_1.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286118_BirA_2.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286118_BirA_3.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286118_BirA_4.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286118_bNanog_1.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286118_bNanog_2.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286118_bNanog_3.CEL |
44.8 Mb |
(ftp)(http) |
CEL |
| GSM286118_bioNanog.bar |
31.7 Mb |
(ftp)(http) |
BAR |
| GSM286118_bioNanog.bed |
112.9 Kb |
(ftp)(http) |
BED |
| Processed data provided as supplementary file |
|
|
|
|
 |
