|
| Status |
Public on Mar 30, 2012 |
| Title |
GenAu MB BCELL v5.0 H3K4me3 Pax5-/- Rag2-/- 142343 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
H3K4me3 ChIP DNA from Pax5-/- Rag2-/- pro-B cells
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Pax5-/- 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).
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| |
|
| Channel 2 |
| Source name |
Input DNA from Pax5-/- Rag2-/- pro-B cells
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Pax5-/- 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 Pax5-/- Rag2-/- pro-B cells H3K4me3
|
| Data processing |
custom R-script
log2(Signal Cy5/Signal Cy3) - tukey.biweight(Signal Cy5/Signal Cy3)
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| |
|
| 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 |
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