 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 10, 2013 |
| Title |
P448_WCE |
| Sample type |
SRA |
| |
|
| Source name |
Diffuse Large B-Cell Lymphoma
|
| Organism |
Homo sapiens |
| Characteristics |
chip antibody: None
antibody catalog number: None - whole cell extract input control
cell type: primary diffuse large B-cell lymphoma tumor
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Primary tissue samples from DLBCL tumors, normal lymph node, or normal tonsil that were flash-frozen and stored in OCT were collected in 10 slices at 20 ¼m. Frozen biopsy specimens of newly diagnosed, previously untreated primary DLBCLs with >80% tumor involvement and known transcriptional subtyping (Monti et al., 2012) were obtained according to Institutional Review Board (IRB)-approved protocols (Brigham & Women Hospital, and Dana-Farber Cancer Institute). A waiver to obtain informed consent was granted by the local IRBs because otherwise discarded tissue was used. After two washes and spin downs in PBS (300g, 5 min), tissue slices were then cross-linked with 1.1% formaldehyde in PBS for 10 min. The formaldehyde was quenched by adding 60 ul/ml of 2.5 M glycine. Glycine and fixative were removed with two washes in cold PBS. Samples were homogenized with a Dounce homogenizer using 10 strokes with pestle A and 10 strokes pestle B in a solution of 10 mM Tris HCl pH8, 10 mM NaCl, 3 mM MgCl2, and 1% NP40. Sample was then transferred to a 15 ml conical tube and rotated in the cold room for 60 min. After this incubation, nuclei were spun down at 1350g for 10 min and transferred to a 3.5 ml tube in 50 mM Tris-HCl pH 7.5, 140 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.7% SDS. Samples were then sonicated on ice for 10 cycles at 30 s (18 W) with 60 s on ice between cycles. All lysis and sonication buffers contain Complete protease inhibitor cocktail (Roche). Sonicated lysates were cleared by centrifuging at 20,000g for 10 min and incubated overnight on a spinning wheel at 4oC with magnetic beads prebound with antibody after diluting sample to 0.1% SDS final. 100 _l of Dynal magnetic beads per sample (Invitrogen) were blocked with 0.5% BSA (w/v) in PBS. Magnetic beads were loaded with 10 _g of each antibody overnight at 4oC. Antibodies used were as follows: Histone H3K27Ac: Abcam ab4729 ; BRD4: Bethyl A301-985A.
Beads were washed three times with sonication buffer, one time with sonication buffer supplemented with 500 mM NaCl, one time with LiCl wash buffer (20 mM Tris pH 8.0, 1 mM EDTA, 250 mMLiCl, 0.5% NP-40, 0.5% Na-deoxycholate) and once with TE. DNA was eluted in elution buffer (50 mM Tris-HCl pH 8, 10mM EDTA, and 1% SDS). Cross-links were reversed overnight at 65oC. RNA and protein were digested with 0.2mg/mL RNase A for two hr followed by 0.2 mg/ml Proteinase K for one hr. DNA was purified with phenol chloroform extraction and ethanol precipitation.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Whole cell extract input control for ChIP
|
| Data processing |
Images analysis and base calling was done using the solexa pipeline.
For all samples reads were aligned to their indicated build using bowtie with parameters -e 70 -k 2 -m 2 -n 2 --best --concise. Seed length (-l) was set to read length for each dataset.
Genome_build: hg18
Supplementary_files_format_and_content: WIG files: For all samples aligned sequences were extended 150bp upstream and 0bp downstream (with respect to read strand) and allocated into 25bp bins. Counts were normalized to reads per million, and bins with at least 1 read per million are shown.
|
| |
|
| Submission date |
Oct 29, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
James Bradner |
| E-mail(s) |
bradner_computation@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Medical Oncology
|
| Lab |
Bradner Lab
|
| Street address |
450 Brookline
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE46663 |
Discovery and Characterization of Super-Enhancer Associated Dependencies in Diffuse Large B-Cell Lymphoma |
|
| Relations |
| BioSample |
SAMN02388193 |
| SRA |
SRX370351 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1254217_P448_WCE.wig.gz |
127.3 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
