|
| Status |
Public on Jun 30, 2012 |
| Title |
H3k36me3_ChIPSeq |
| Sample type |
SRA |
| |
|
| Source name |
H3k36me3_ChIPSeq
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Spleen-derived Spi-1-transgenic erythroleukemic cells
strain: DBA2J
chip antibody: H3K36me3
|
| Treatment protocol |
No treatment
|
| Growth protocol |
cells were grown in a-MEM medium supplemented with 5% FBS and erythropoetin (Epo) (1U/mL)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
100ng ChIP (or input) DNA was combined with 25mg of 2 microns borosilicate glass dry spheres (Duke Scientific) in round bottom borosilicate tubes. The DNA was re-sheared using the Covaris S2 Sample Prep System (Covaris) for 10 min at 4°C with maximum intensity settings to generate small DNA fragments in the 90-bp range. Sheared DNA was purified using the MinElute reaction cleanup kit (Qiagen). The End-It DNA end-repair kit (Epicentre) was used to blunt the ends of the sheared DNA fragments. The blunted DNA fragments were purified using the MinElute Reaction Cleanup Kit (Qiagen). Sheared DNA was quantified using a Qubit fluorometer (Invitrogen) and P1 and P2 adapters (Applied Biosystems) were ligated to the DNA for 10 min at room temperature using Quick ligase enzyme (NEB). Excess free adaptors were removed using the AMPure PCR purification system (Agencourt). Adapted DNA fragments were size selected by running samples through a 6% DNA retardation PAGE gel (Invitrogen). DNA fragments 150-200 bp were specifically selected using a scalpel, and the gel fragment shredded by centrifugation through a hole made with a 21G needle. ChIP fragment libraries were amplified using library PCR primers for the P1 and P2 adapters to amplify correctly adapted fragments (containing P1 and P2 on opposing ends) from a shredded gel suspension made by mixing the shredded gel with PCR SuperMix High Fidelity (Invitrogen). PCR was performed for just enough cycles (~13) to generate visible DNA on ethidium-stained agarose gels. Library DNA sample quality and size were verified using a Bioanalyzer (Agilent).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD System 2.0 |
| |
|
| Data processing |
SOLiD ChIP-seq DNA fragment libraries were sequenced by SOLiD system 2.0 and SOLiD system 3.0 chemistries to produce DNA sequence reads of 35 and 50 nucleotides, respectively. Sequencing reads were aligned to the mm9 mouse genome using the SOLiD pipeline tools (CoronaLite, Applied Biosystems) with up to three and five mismatches per read for SOLiD system 2.0 and system 3.0, respectively. Only the reads with a unique position in the genome were kept for the following analysis. Spi-1 reads from ChIP-seq of two individual mice were pooled. To detect peaks corresponding to putative binding regions, we used FindPeaks algorithm (Fejes et al. 2008) and the MICSA pipeline (Boeva et al. 2010).
genome build: mm9
|
| |
|
| Submission date |
Nov 10, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Valentina Boeva |
| Organization name |
Institut Cochin
|
| Department |
Inserm U1016
|
| Street address |
24 rue du Faubourg Saint-Jacques
|
| City |
Paris |
| ZIP/Postal code |
75014 |
| Country |
France |
| |
|
| Platform ID |
GPL10279 |
| Series (1) |
| GSE33611 |
Spi-1/PU.1 activates transcription through clustered DNA occupancy in erythroleukemia |
|
| Relations |
| SRA |
SRX105207 |
| BioSample |
SAMN00752425 |
