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| Status |
Public on Nov 27, 2012 |
| Title |
AF4_HiSeq_ChIPSeq |
| Sample type |
SRA |
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| Source name |
Acute lymphoblastic leukemia cells with MLL/AF4 translocation, AF4 ChIP
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| Organism |
Homo sapiens |
| Characteristics |
cell type: RS4;11 ALL cells
chip antibody: AF4
chip antibody host: rabbit
antibody vendor: Abcam
antibody catalog #: ab31812
antibody lot#: GR33890-1
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| Growth protocol |
The human RS4;11 ALL cells were maintained in Roswell Park Memorial Institute medium (RPMI-1640, Invitrogen, CA) with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100 μg/ml streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP: For histone ChIP, RS4;11 cells were fixed with 1% formaldehyde, lysed and sonicated to generate fragments less than 500bp. For MLL and AF4 ChIP, RS4;11 cells were double-fixed with 2mM Disuccinimidyl Glutarate and then 1% formaldehyde, lysed and sonicated to generate fragments less than 500bp. Sonicated lysates were incubated with antibodies overnight, and after increasing stringency washes, immunocomplexes were recovered and DNA was isolated. The ChIP protocol in RS4;11 cells was followed as outlined in Milne et al. (PMID 19277579), using a formaldehyde fixation protocol for H3K79me2 and H3K4Me3, and a 45 minute, 2mMDSG and a 30 minute 1% double-fixation protocol for MLLN and AF4C.
Library construction: Genomic DNA-fragment libraries were prepared using the Illumina ChIP-seq Library Preparation Kit following the manufacturer's instructions (Illumina, CA). Briefly, 10ng of purified ChIP DNA was end repaired by conversion of overhangs into phosphorylated blunt ends with the use of T4 DNA polymerase and E. coli DNA polymerase I Klenow fragment. Illumina single-end adapters were ligated to the ends of the DNA fragments. Ligation products were purified on a 2% agarose gel with a size selection of 200-300bp. Fifteen PCR cycles were performed with Illumina genomic DNA primers that anneal to the ends of the adapters. The purified PCR-amplified fragment libraries were quantified with the use of the PicoGreen dsDNA Quantitation Assay with the Qubit Fluorometer (Invitrogen, CA). The size range of libraries was validated on the Agilent Technologies 2100 Bioanalyzer with the High Sensitivity DNA Kit (Agilent, CA).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Counts: Raw image data were converted into base calls via the Illumina pipeline CASAVA version 1.7 with default parameters. All 36-bp-long reads were mapped to the reference human genome sequence, hg18, using Illumina’s ELAND aligner with the default parameters. Only reads mapping uniquely to the genome with not more than 2 mismatches were retained for downstream analysis.
Peaks: Peak detection was performed with the ChIPseeqer program (Giannopoulou and Elemento, BMC Bioinformatics 2011, 12:277 (PMID 21736739)) and annotated to genes and/or promoters based on hg18 RefSeq genes downloaded from the UCSC Genome Browser.
Genome_build: hg18
Supplementary_files_format_and_content: BIGWIG: Counts.
Supplementary_files_format_and_content: BED: Peaks.
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| Submission date |
Jun 01, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Huimin Geng |
| E-mail(s) |
huimin.geng@ucsf.edu
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| Organization name |
UCSF
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| Department |
Department of Laboratory Medicine
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| Street address |
513 Parnassus Ave., MSB S-1480
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE34941 |
Integrative Epigenomic Analysis Identifies Biomarkers and Therapeutic Targets in Adult B-Acute Lymphoblastic Leukemia |
| GSE38403 |
Integrative Epigenomic Analysis Identifies Biomarkers and Therapeutic Targets in Adult B-Acute Lymphoblastic Leukemia [ChIP-seq] |
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| Relations |
| SRA |
SRX151223 |
| BioSample |
SAMN01001960 |
| Named Annotation |
GSM941544_s_2_AF4_HiSeq.nh.overlap.bigWIG |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM941544_AF4_HiSeq_hg18_targets_eland.peak.bed.gz |
50.2 Kb |
(ftp)(http) |
BED |
| GSM941544_s_2_AF4_HiSeq.nh.overlap.bigWIG |
703.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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