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Sample GSM1051313 Query DataSets for GSM1051313
Status Public on Aug 14, 2013
Title RAG1KO H3K27ac
Sample type genomic
 
Channel 1
Source name H3K27ac (abcam-ab4729) ChIP DNA from Rag1-/- DN cells
Organism Mus musculus
Characteristics genotype: Rag1KO
cell type: DN cells
strain: C57BL/6J
chip antibody: H3K27ac
antibody vendor/catalog: abcam-ab4729
Treatment protocol Rag1KO DN cells were obtained from thymus of 3-4 week old mice.
Extracted molecule genomic DNA
Extraction protocol 5 million cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 400 bp using a BiorupterTM sonicator (Diagenode). 5 µg of antibody was incubated with Dynabeads ProteinA beads (Invitrogen at 4C for 2-4 hours . The chromatin (from 5 million cells) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and added to the anitbody conugated to the Dynabeads overnight at 4C. After incubation, the immune complexes were collected by centrifugation and washed with the following buffers each for 3 min at 4C;low salt (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and TE (10 mM Tris pH8.0, 1 mM EDTA). The protein-DNA complexes were eluted from the beads in 250 µl elution buffer (1% SDS, 100 mM NaHCO3) at 65C overnight followed by the addition of proteinaseK to 500 µg/ml and incubaton at 56C for 1 hour. Genomic DNA was isolated using QIAquick PCR purification column and subject to whole genome amplication (Sigma).
Label Cy5
Label protocol 1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
 
Channel 2
Source name Input DNA from Rag1KO DN cells
Organism Mus musculus
Characteristics genotype: Rag1KO
cell type: DN cells
strain: C57BL/6J
chip antibody: none, input DNA
Treatment protocol Rag1KO DN cells were obtained from thymus of 3-4 week old mice.
Extracted molecule genomic DNA
Extraction protocol 5 million cells were fixed with 1% formaldehyde in culture medium for 10 min at room temperature followed by quenching with 0.125 M glycine for 5 min. The cells were washed twice with ice-cold PBS and lysed in 1 ml of lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH8.0) for at least 30 min on ice. The crosslinked chromatin was sheared to an average size of 400 bp using a BiorupterTM sonicator (Diagenode). 5 µg of antibody was incubated with Dynabeads ProteinA beads (Invitrogen at 4C for 2-4 hours . The chromatin (from 5 million cells) was diluted 10-fold in dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA 16.7 mM Tris pH8.10, 167 mM NaCl) and added to the anitbody conugated to the Dynabeads overnight at 4C. After incubation, the immune complexes were collected by centrifugation and washed with the following buffers each for 3 min at 4C;low salt (150 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), high-salt buffer (500 mM NaCl, 50 mM Tris pH8.0, 0.1% SDS), LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and TE (10 mM Tris pH8.0, 1 mM EDTA). The protein-DNA complexes were eluted from the beads in 250 µl elution buffer (1% SDS, 100 mM NaHCO3) at 65C overnight followed by the addition of proteinaseK to 500 µg/ml and incubaton at 56C for 1 hour. Genomic DNA was isolated using QIAquick PCR purification column and subject to whole genome amplication (Sigma).
Label Cy3
Label protocol 1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy3 nonamers (ChIP DNA isolated from normal rabbit IgG-immunoprecipitated cells) per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
 
 
Hybridization protocol The labeled ChIP DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in 42C 0.2% SDS/0.2x SSC, room temperature 0.2x SSC, and 0.05x SSC. Hybridization buffers and washes were completed using manufacturer's protocols (http://www.nimblegen.com/products/lit/lit.html)
Scan protocol Arrays were scanned on an Axon 4000B scanner per manufacturer's protocol (http://www.nimblegen.com/products/lit/lit.html).
Description Chip-chip Rag1KO DN cells -H3K27ac
Data processing Arrays were processed using Nimblegen's standard protocol for Nimblescan 2.4 ChIP data extraction.
 
Submission date Dec 11, 2012
Last update date Aug 14, 2013
Contact name Eugene M Oltz
Organization name Washington Univeristy in St.Louis
Department Pathology and Immunology
Street address 660 S Euclid Avenue
City St.Louis
State/province MO
ZIP/Postal code 63110
Country USA
 
Platform ID GPL16370
Series (2)
GSE42851 A Unifying Model for Molecular Determinants of the Pre-selection Vβ Repertoire [ChIP-chip]
GSE49234 A Unifying Model for Molecular Determinants of the Pre-selection Vb Repertoire

Data table header descriptions
ID_REF
VALUE scaled, log2 (ChIP/Input) ratio

Data table
ID_REF VALUE
CHR12FS111666845 0.4
CHR12FS111666860 0.28
CHR12FS111666880 0.43
CHR12FS111666905 0.24
CHR12FS111666920 0.2
CHR12FS111666940 0.24
CHR12FS111666960 0.4
CHR12FS111666975 0.12
CHR12FS111666990 0.19
CHR12FS111667015 0.62
CHR12FS111667035 0.55
CHR12FS111667055 0.31
CHR12FS111667070 0.52
CHR12FS111667085 0.16
CHR12FS111667105 -0.04
CHR12FS111667125 0.05
CHR12FS111667150 0.04
CHR12FS111667170 -0.16
CHR12FS111667190 -0.1
CHR12FS111667200 -0.3

Total number of rows: 690279

Table truncated, full table size 33267 Kbytes.




Supplementary file Size Download File type/resource
GSM1051313_DN_H3K27ac_Cy3.pair.gz 11.5 Mb (ftp)(http) PAIR
GSM1051313_DN_H3K27ac_Cy5.pair.gz 11.4 Mb (ftp)(http) PAIR
Processed data included within Sample table
Processed data provided as supplementary file

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