 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 31, 2013 |
| Title |
RUNX1_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
Acute lymphoblastic leukemia cells with MLL/AF4 translocation
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: SEM
chip antibody: Runx1
chip antibody host, amount, catelog number and provider: Rabbit, 2ug, ab23980, Abcam
|
| Growth protocol |
The human SEM ALL cells were maintained in Iscove's Modified Dulbecco's medium (IMDM, Invitrogen, CA) with 10% fetal bovine serum (FBS).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
SEM 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 SEM cells was followed as previously outlined (Milne et al., Methods Mol Biol 2009, 538: 409-423), using a 45 minute, 2mM DSG and a 45 minute 1% double fixation protocol.
Genomic DNA-fragment libraries were prepared using the Illumina ChIP-seq Library preparation Kit following the manufacture’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).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
NA
|
| Data processing |
Counts: Raw image data were converted into base calls via the Illumina pipeline RTA version 1.12.4. All 50-bp-long paired-end reads were mapped to the reference human genome sequence, hg18, using BWA aligner with the default parameters (Li and Durbin, Bioinformatics 2009, 25:1754-60). 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) and annotated to genes and/or promoters based on hg18 refseq genes downloaded from the UCSC Genome Browser
Genome_build: hg18
|
| |
|
| Submission date |
Nov 06, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Erica Ballabio |
| E-mail(s) |
erica.ballabio@imm.ox.ac.uk
|
| Phone |
0044 1865 222418
|
| Organization name |
Univeristy of Oxford
|
| Department |
The Weatherall Institute of Molecular Medicine
|
| Lab |
Tom Milne
|
| Street address |
headley way
|
| City |
Oxford |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL11154 |
| Series (1) |
| GSE42075 |
RUNX1 is a key target gene in t(4;11) leukemias and contributes to gene activation by interacting with the AF4-MLL complex |
|
| Relations |
| SRA |
SRX202967 |
| BioSample |
SAMN01801716 |
| Named Annotation |
GSM1032075_SEM_RUNX1.bigWIG |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1032075_SEM_RUNX1.bigWIG |
312.4 Mb |
(ftp)(http) |
BIGWIG |
| GSM1032075_SEM_RUNX1_target_1.bigBED |
665.6 Kb |
(ftp)(http) |
BIGBED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
|
|
|
|
 |
