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Sample GSM516404 Query DataSets for GSM516404
Status Public on Oct 14, 2010
Title bioMax
Sample type genomic
 
Channel 1
Source name bioMax ChIP DNA from mouse ES cells
Organism Mus musculus
Characteristics cell type: bioMax J1 mES cells
fraction: ChIP
target: Max
strain: 129S4/SvJae
Treatment protocol Normal J1 ES growth condition
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 expressing BirA
Organism Mus musculus
Characteristics cell type: BirA expressing J1 mES cells
fraction: reference (mock ChIP)
strain: 129S4/SvJae
Treatment protocol Normal J1 ES growth condition
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 n/a
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. BAR and BED files were generated by using Mm_PromPR_v02-1_NCBIv36.NR.bpmap.
 
Submission date Feb 26, 2010
Last update date Oct 14, 2010
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)
GSE20551 ChIP-chip from mouse embryonic stem (mES) cells

Supplementary file Size Download File type/resource
GSM516404_BirA_1.CEL.gz 17.3 Mb (ftp)(http) CEL
GSM516404_BirA_2.CEL.gz 17.5 Mb (ftp)(http) CEL
GSM516404_BirA_3.CEL.gz 18.6 Mb (ftp)(http) CEL
GSM516404_BirA_4.CEL.gz 18.7 Mb (ftp)(http) CEL
GSM516404_bMax_1.CEL.gz 20.3 Mb (ftp)(http) CEL
GSM516404_bMax_2.CEL.gz 20.2 Mb (ftp)(http) CEL
GSM516404_bMax_3.CEL.gz 20.2 Mb (ftp)(http) CEL
GSM516404_bioMax.bar.gz 22.6 Mb (ftp)(http) BAR
GSM516404_bioMax.bed.gz 74.2 Kb (ftp)(http) BED
Processed data provided as supplementary file

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