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Sample GSM701440 Query DataSets for GSM701440
Status Public on Mar 30, 2012
Title GenAu MB BCELL v5.0 H3K4me3 Rag2-/- 142355
Sample type genomic
 
Channel 1
Source name H3K4me3 ChIP DNA from Rag2-/- pro-B cells
Organism Mus musculus
Characteristics genotype/variation: Rag2-/-
age: 5 days in culture
cell type: pro-B cells
strain: C57BL/6J
antibody: H3K4me3
type: ChIP
amplification: wga
antibody vendor: Upstate
antibody lot#: 07-473
Treatment protocol pro-B cells were MACs sorted from the bone marrow of 3-5 week old mice.
Growth protocol pro-B cells were cultured for 5 days in the presence of IL-7 on OP9 feeder cells.
Extracted molecule genomic DNA
Extraction protocol 5x10E6 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 500 bp by ten 30-second pulses using a BiorupterTM sonicator (Diagenode). The lysate was then centrifuged to remove cell debris, and the chromatin was quantified in a UV spectrophotometer. The chromatin (400 µg) 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 pre-cleared with 25 µl protein A sepharose beads (pre-absorbed with salmon sperm DNA and BSA) for 2 h at 4C. The chromatin supernatant was then incubated overnight with 5 µg of purified antibody at 4C. After incubation with protein A sepharose beads (50 µl) for 2h at 4C, the immune complexes were collected by centrifugation and washed with the following buffers each for 10 min at 4C; RIPA buffer (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, 1% NP40) LiCl buffer (250 mM LiCl, 50 mM Tris pH8.0, 0.5% Na deoxycholate, 1% NP40) and 2x TE (20 mM Tris pH8.0, 2 mM EDTA). The protein-DNA complexes were eluted from the beads in 450 µl elution buffer (1% SDS, 100 mM NaHCO3) at 55C for 2 h followed by the addition of proteinaseK to 500 µg/ml and overnight incubaton at 65C. Genomic DNA was isolated from the precipitated material as well as from the sheared chromatin input (1/100 of the material used for ChIP) by phenol extraction and ethanol precipitation.
Label Cy5
Label protocol 1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy5 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 Rag2-/- pro-B cells
Organism Mus musculus
Characteristics genotype/variation: Rag2-/-
age: 5 days in culture
cell type: pro-B cells
strain: C57BL/6J
antibody: none
type: Input
Treatment protocol see channel 1
Growth protocol see channel 1
Extracted molecule genomic DNA
Extraction protocol see channel 1
Label Cy3
Label protocol 1 µg ChIP DNA was directly labeled by Klenow (New England Biolabs) random priming with Cy5 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 Rag2-/- pro-B cells H3K4me3
Data processing custom R-script
log2(Signal Cy5/Signal Cy3) - tukey.biweight(Signal Cy5/Signal Cy3)
 
Submission date Apr 04, 2011
Last update date Mar 30, 2012
Contact name Meinrad Busslinger
E-mail(s) busslinger@imp.ac.at
Phone 00431-79730
Organization name Instutute of Molecular Pathology
Street address Dr. Bohrgasse 7
City Vienna
ZIP/Postal code 1030
Country Austria
 
Platform ID GPL13123
Series (1)
GSE28373 The transcription factor Pax5 regulates its target genes by recruiting chromatin-modifying proteins in committed B cells

Data table header descriptions
ID_REF
Cy3 Cy3 signal, 532nm
Cy5 Cy5 signal, 635nm
VALUE log2(Signal Cy5/Signal Cy3) - tukey.biweight(Signal Cy5/Signal Cy3)

Data table
ID_REF Cy3 Cy5 VALUE
6006_0001_0001 255.56 432.44 0.24
6006_0001_0085 null null null
6006_0001_0087 null null null
6006_0001_0089 null null null
6006_0001_0091 null null null
6006_0001_0093 null null null
6006_0001_0095 null null null
6006_0001_0097 null null null
6006_0001_0099 null null null
6006_0001_0101 null null null
6006_0001_0103 null null null
6006_0001_0105 null null null
6006_0001_0107 null null null
6006_0001_0109 null null null
6006_0001_0111 null null null
6006_0001_0113 null null null
6006_0001_0115 null null null
6006_0001_0117 null null null
6006_0001_0119 null null null
6006_0001_0121 null null null

Total number of rows: 392778

Table truncated, full table size 13055 Kbytes.




Supplementary file Size Download File type/resource
GSM701440_142355_532.pair.gz 6.4 Mb (ftp)(http) PAIR
GSM701440_142355_635.pair.gz 6.5 Mb (ftp)(http) PAIR
Processed data included within Sample table

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