|
| Status |
Public on Mar 27, 2012 |
| Title |
RUNX1ETO_siRE_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
Kasumi-1 transfection with RUNX1/ETO siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Kasumi-1
target: RUNX1/ETO
antibody: ETO(C20)sc (lot number F1809)
|
| Treatment protocol |
Kasumi-1 cells were transfected with the 200 nM of siRNA using a Fischer EPI 3500 electroporator (Fischer, Heidelberg, Germany) as described previously (Martinez et al., 2004). The following siRNAs were used: RUNX1/ETO siRNAs (sense, 5'-CCU CGA AAU CGU ACU GAG AAG-3'; antisense, 5'-UCU CAG UAC GAU UUC GAG GUU-3')
|
| Growth protocol |
The Kasumi-1 cell line (acute myeloid leukemia FAB M2 with t(8;21)) was obtained from the DSMZ cell line repository (http://www.dsmz.de/ mutz/mutzhome.htm). The cells were grown in RPMI 1640 medium (Invitrogen, Heidelberg, Germany) supplemented 2 mM L-glutamine with 10% FCS and maintained in a humidified 37C incubator with 5% CO.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Libraries of DNA fragments from chromatin immunoprecipitation or DNase I treatment were prepared from approximately 10ng of DNA. Firstly, overhangs were repaired by treatment of sample material with T4 DNA polymerase, T4 PNK and Klenow DNA polymerase (all enzymes obtained from New England Biolabs UK) in a reaction also containing 50 mM Tris-HCl,10 mM MgCl2, 10 mM Dithiothreitol, 0.4 mM dNTPs and 1 mM ATP. Samples were purified after each step using Qiagen MinElute columns (according to the manufacturer's guidelines). "A" bases were added to 3' ends of fragments using Klenow Fragment (3' - 5' exo- ), allowing for subsequent ligation of adapter oligonucleotides (Illumina part #1000521) using Quick T4 DNA ligase. After a further column clean up to remove excess adaptors, fragments were amplified in an 18 cycle PCR reaction using adapter-specific primers The libraries were purified and adapter dimers removed by running PCR products on 2% agarose gels and excising gel slices corresponding to fragments approximately 200-300 bp in size, which were then extracted using the Qiagen gel extraction kit. Once prepared, DNA libraries were subject to massively parallel DNA sequencing on an Illumina Genome Analyzer.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Data processing |
Peaks: Peak finding analysis was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/) using hg18
|
| |
|
| Submission date |
May 11, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Salam Adli Assi |
| E-mail(s) |
s.a.assi@bham.ac.uk
|
| Organization name |
University of Birmingham
|
| Department |
Institute for Cancer and Genomic Sciences
|
| Street address |
IBR
|
| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL9052 |
| Series (2) |
| GSE29222 |
Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding [ChIP-Seq and DNAse-Hypersensitivity data] |
| GSE29225 |
Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding |
|
| Relations |
| SRA |
SRX062338 |
| BioSample |
SAMN00265092 |
