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| Status |
Public on Jan 28, 2013 |
| Title |
Erythroid GATA1 |
| Sample type |
SRA |
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| Source name |
R3/R4 Erythroid cells
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| Organism |
Homo sapiens |
| Characteristics |
cell: R3/R4 Erythroid cells
chip antibody: GATA1
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| Treatment protocol |
ChIP assays were performed as previously described with minor variations (Steiner, L. A., Maksimova, Y., Schulz, V., Wong, C., Raha, D., Mahajan, M. C., Weissman, S. M., and Gallagher, P. G. (2009) Mol Cell Biol). 1 × 107 cells were cross-linked with 1% formaldehyde for 10 min at room temperature, followed by Dounce homogenization. Cross-linked nuclei were isolated, followed by sonication with a Covaris S2 shearing device (duty cycle 10%, intensity 5, 15 minutes), to obtain chromatin-containing DNA fragments with an average size of ~200-400bp. For each ChIP, 20 µg of antibody or the appropriate control IgG species was used. Samples were immunoprecipitated with antibodies against GATA1 (sc-265; Santa Cruz), NF-E2 (sc-22827; Santa Cruz), KLF1 (ab2483; Abcam), SCL/Tal1 (sc-12984; Santa Cruz), P300 (sc-585; Santa Cruz) and nonspecific rabbit IgG (sc-2091; Santa Cruz). The antigen-antibody complex was captured on protein G beads, washed four times with radioimmunoprecipitation assay buffer, and then washed with phosphate-buffered saline. The DNA-protein complex was eluted from the protein G beads with 1% sodium dodecyl sulfate at 65°C, and cross-linking of DNA-protein adducts was reversed by 4 hours of incubation at 65°C. After proteinase K and RNase digestion of the reverse-cross-linked sample, DNA was cleaned with the QIAquick PCR Purification Kit (Qiagen) according to manufacturer instructions.
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| Growth protocol |
Human CD34+-selected stem and progenitor cells were obtained from the Yale Center for Excellence in Molecular Hematology. To obtain primary human erythroid cells, CD34+ cells were cultured and selected as described.(Panzenbock, B., Bartunek, P., Mapara, M. Y., and Zenke, M. (1998) Blood 92, 3658-3668. Migliaccio, A. R., Migliaccio, G., Di Baldassarre, A., and Eddleman, K. (2002) Bone Marrow Transplant 30, 75-80) Briefly, Human CD34-selected stem and progenitor cells were cultured for 9 to 14 days in StemSpan SF expansion medium which contains human transferrin (200 ng/ml) and insulin (10 ng/ml) (StemSpan 09650) and was supplemented with estradiol (1 micromolar), dexamethasone (1 micromolar), Flt3 ligand (100 ng/ml), stem cell factor (100 ng/ml), interleukin-3 (50 ng/ml), interleukin-6 (20 ng/ml), insulin-like growth factor 1 (50 ng/ml), and erythropoietin (3 U/ml). FACS analysis was used to analyze cellular expression of CD71 (transferrin receptor) and CD235a (glycophorin A). These cells represent the R3/R4 cell population of nucleated erythroid cells defined by Zhang et al.(Zhang, J., Socolovsky, M., Gross, A. W., and Lodish, H. F. (2003) Blood 102, 3938-3946)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 500ng of ChIP DNA was run on a 1.5% agarose gel to size select ChIP DNA in the 200-300-bp range. The size-selected ChIP DNA was purified using a gel extraction kit (Qiagen) and processed for sequencing as per manufacturer's protocol. Briefly, size-selected ChIP DNA was end repaired using an End-IT DNA end repair kit (ER0720; Epicenter), followed by addition of an adenine base at the 3′ end by Klenow reaction and Solexa adaptor ligation. The modified DNA was then PCR amplified with one initial heating step of 98°C for 30 s, followed by 15 cycles of amplification with a melting temperature of 98°C for 30 s, an annealing temperature of 65°C for 30 s, and a product extension at 72°C for 30 s. At the end of the amplification, a final extension at 72°C for 5 min was performed. Amplified ChIP DNA was then size selected on a 1.5% agarose gel, purified by using a gel extraction kit (Qiagen), and subjected to deep sequencing.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Basecalls were performed using CASAVA version 1.7.0
ChIP-seq reads were aligned to the hg18 genome assembly using elandv2.
The eland extended format alignments were converted to bed format using the elandextended2bed.py script.
Peaks were called against the total input sample using macs version 1.4.0rc2 with default parameters.
Quality scores in the fastq files were converted from Illumina1.3 to sanger using the fq_all2std.pl script.
Genome_build: hg18
Supplementary_files_format_and_content: The macs peaks.bed output files have a fifth field that is -10*log10(pvalue)
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| Submission date |
Jan 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Vince Schulz |
| E-mail(s) |
vincent.schulz@yale.edu
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| Organization name |
Yale University
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| Department |
Department of Pediatrics
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| Lab |
Gallagher
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| Street address |
333 Cedar St. LMP 4085
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06519 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (2) |
| GSE43625 |
Identification of Biologically Relevant Enhancers in Human Erythroid Cells [ChIP-Seq] |
| GSE43626 |
Identification of Biologically Relevant Enhancers in Human Erythroid Cells |
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| Relations |
| Reanalyzed by |
GSE59801 |
| SRA |
SRX218418 |
| BioSample |
SAMN01889554 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1067274_Erythroid_GATA1_peaks.bed.gz |
616.5 Kb |
(ftp)(http) |
BED |
| GSM1067274_Erythroid_GATA1_reads.bed.gz |
288.5 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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