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| Status |
Public on Nov 04, 2008 |
| Title |
Normal_preB_H3K79 (3 samples) |
| Sample type |
genomic |
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| Source name |
H3K79me2 ChIP of normal preB cells
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| Organism |
Mus musculus |
| Characteristics |
Purified normal bone marrow B220+ CD43- IgM- IgD- cells
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
3-10×10e5 cells were used. Briefly, cells were cross-linked by addition of 1% formaldehyde for 10 min at room temp. Cross-linking was terminated by adding final 125 mM glycine and cells were washed with cold PBS containing PMSF, collected by centrifugation and washed in PBS twice. The pellet was resuspended in SDS buffer (50 mM Tris-Cl pH 8.1; 100 mM NaCl; 0.5% SDS; 5 mM EDTA; 0.2% NaN3 and protease inhibitors) and snap frozen. After thawing the nuclear pellets were collected by centrifugation and resuspended in ChIP buffer (50mM Tris 8.1; 100mM NaCl; 0.3% SDS; 1.5% Triton X-100; 5 mM EDTA; 0.2% NaN3; and protease inhibitors). Cells were sonicated to fragment DNA to an average size of 0.5-1 kb. The sample was centrifuged at 10000g at 4° C for 10 min, the supernatant collected and pre-cleared with protein-A beads at 4°C for 2 hrs. The sample was then incubated with anti-dimethyl H3K79 antibody (ab# ab3594) and protein-A beads at 4°C overnight. Immunoprecipitated protein-DNA complexes were sequentially washed with Mixed Micelle buffer (20 mM Tris pH 8.1; 150mM NaCl; 5mM EDTA; 0.5% SDS; 1% Triton X-100; 6% Sucrose), buffer 500 (0.1% Deoxycholate, 1% Triton X-100, 1 mM EDTA, 50 mM HEPES pH 7.5, 500 mM NaCl), LiCl buffer (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 5 min at 4C. Protein-DNA complexes were eluted in TE with 1%SDS buffer supplemented with proteinase K (500u) for 3 hrs at 55oC. The sample was treated with RNase A and DNA fragments were purified using Qiagen's PCR purification kit, and eluted in 100 ul of TE buffer.
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| Label |
Biotin
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| Label protocol |
Each ChIP was amplified using ligation-mediated PCR (LM-PCR). Labeling was according to standard Affymetrix protocols.
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| Hybridization protocol |
3 micrograms of labelled material was hybridized according to the standard Affymetrix protocol.
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| Scan protocol |
Standard Affymetrix protocol
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| Description |
normal preB H3K79 CHIP: preB_1_MmProm.CEL, preB_3_MmProm.CEL, preB_3_MmProm.CEL
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| Data processing |
Regions of above-background signal were identified by Model-based Analysis of Tiling array (MAT) software (http://chip.dfci.harvard.edu/~wli/MAT/) run with bandwidth = 300, max gap = 300, minimum probes = 10 and p-value cutoff = 1x10e-5 and using a bpmap for mouse NCBI genome build 36 (Mm8) downloaded from the MAT website.
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| Submission date |
Aug 06, 2008 |
| Last update date |
Nov 04, 2008 |
| Contact name |
Madeleine E. Lemieux |
| E-mail(s) |
mlemieux@bioinfo.ca
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| Phone |
617-595-6695
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| URL |
http://www.bioinfo.ca
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| Organization name |
Bioinfo
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| Street address |
275 Main St., Suite 252
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| City |
Plantagenet |
| State/province |
ON |
| ZIP/Postal code |
K0B 1L0 |
| Country |
Canada |
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| Platform ID |
GPL5811 |
| Series (2) |
| GSE12361 |
Genome-wide analysis of H3K79 dimethylation in normal and MLL-AF4 leukemic pre-B cells |
| GSE12363 |
H3K79 methylation profiles define murine and human MLL-AF4 leukemias |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM310416_preB_1_MmProm.CEL.gz |
19.1 Mb |
(ftp)(http) |
CEL |
| GSM310416_preB_2_MmProm.CEL.gz |
19.0 Mb |
(ftp)(http) |
CEL |
| GSM310416_preB_3_MmProm.CEL.gz |
19.4 Mb |
(ftp)(http) |
CEL |
| GSM310416_set1_preB.Mm_PromPR_v02-1_NCBIv36.NR.bpmap_matscore.bar.gz |
22.6 Mb |
(ftp)(http) |
BAR |
| GSM310416_set1_preB.bed.gz |
121.2 Kb |
(ftp)(http) |
BED |
| Processed data provided as supplementary file |
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