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Sample GSM286123 Query DataSets for GSM286123
Status Public on May 06, 2008
Title bioZfp281
Sample type genomic
 
Channel 1
Source name bioZfp281 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
bZfp281_1.CEL
bZfp281_2.CEL
bZfp281_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
GSM286123_BirA_1.CEL 44.8 Mb (ftp)(http) CEL
GSM286123_BirA_2.CEL 44.8 Mb (ftp)(http) CEL
GSM286123_BirA_3.CEL 44.8 Mb (ftp)(http) CEL
GSM286123_BirA_4.CEL 44.8 Mb (ftp)(http) CEL
GSM286123_bZfp281_1.CEL 44.8 Mb (ftp)(http) CEL
GSM286123_bZfp281_2.CEL 44.8 Mb (ftp)(http) CEL
GSM286123_bZfp281_3.CEL 44.8 Mb (ftp)(http) CEL
GSM286123_bioZfp281.bar 31.7 Mb (ftp)(http) BAR
GSM286123_bioZfp281.bed 42.7 Kb (ftp)(http) BED
Processed data provided as supplementary file

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