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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 23, 2012 |
| Title |
HoxB4 ChIP_day 16 |
| Sample type |
SRA |
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| Source name |
EB cells cultured with a hematopietic cytokine cocktail for 10 days
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| Organism |
Mus musculus |
| Characteristics |
cell type: EB cells cultured with a hematopietic cytokine cocktail for 10 days
chip antibody: anti-HoxB4
chip antibody vendor: Epitomics
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| Growth protocol |
To differentiate ES cells into HSCs, a 2-stage culture strategy was adopted. Initially, ES cells were subjected to embryoid body (EB) formation over 6 days. At this stage, ES cells were plated to an ultra-low attachment Petri dish at a concentration of 2000 cells/mL in a methycellulose-based differentiation medium containing Iscove modified Dulbecco medium (Invitrogen), 15% FCS, 300 μg/mL transferrin, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin, 5% protein-free hybridoma medium (Invitrogen), 4x10^-4 M monothioglycerol, and 50 μg/mL ascorbic acid. At second stage, EBs were dissociated into single-cell suspension using trypsin (2.75%), and 3.5x10^6 cells/mL cells were re-plated on another ultra-low attachment Petri dish in a serum-free hematopoietic differentiation medium that contained StemPro34 plus nutrient supplement (Invitrogen) and a cocktail of hematopoietic cytokines including murine stem cell factor (mSCF; 100 ng/mL; R&D Systems, Minneapolis, MN), mIL-3 (2 ng/mL), mIL-6 (5 ng/mL), Flt3-L (10 ng/mL), insulin-like growth factor-1 (IGF-1; 40 ng/mL; Promega, Madison, WI), and dexamethasone (1 μM; Sigma-Aldrich, St Louis, MO). Culture medium was changed every other day and cell density maintained below 4x106 cells/mL.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin fragments with HoxB4 binding sites were enriched by immunoprecipitation with a rabbit monoclonal antibody against human HoxB4 immunogen (Epitomics, Burlingame, CA). After reversal of cross-linking, the enriched DNA fragments were precipitated and further purified using a PCR clean-up kit (Qiagen). Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Sample 3
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| Data processing |
D16_HoxB4_IP.counts.wig; genome build: mm9
Counts: Sequence reads were obtained and mapped to the mouse (July, 2007) genomes using the Bowtie algorithm. Uniquely mapped reads with two or fewer mismatches were retained and read starts were summed in sliding windows of 200 bp to create summary windows. Counts included.
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| Submission date |
Nov 29, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Kai Tan |
| Organization name |
University of California San Diego
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| Department |
Bioengineering
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| Street address |
9500 Gilman Drive
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (2) |
| GSE34013 |
Time-course HoxB4 ChIP-Seq during HSC development from ES cells |
| GSE34014 |
Gene expression profiling and ChIP-Seq study of HoxB4-mediated HSC development from ES cells |
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| Relations |
| SRA |
SRX109457 |
| BioSample |
SAMN00760890 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM840468_D16_HoxB4_IP.counts.wig.gz |
31.2 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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