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| Status |
Public on Jun 27, 2016 |
| Title |
HSPC_CTCF_2 |
| Sample type |
SRA |
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| Source name |
Human Stem and Progenitor Cell
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HSPC
chip antibody: CTCF
antibody source: Active Motif, DMABT-H19813
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| Growth protocol |
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 in StemSpan SF expansion medium (StemSpan 09650) with estradiol (100 ng/ml), dexamethasone (10 ng/ml), human transferrin (200 ng/ml), insulin (10 ng/ml), 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) for 9 to 14 days. 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 |
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 was used. Samples were immunoprecipitated with antibodies against antibody against CTCF (Millipore,07-729), the SA-1 subunit of cohesin (Abcam, ab4457), trimethyl histone 3 lysine 27 (Abcam ab6002) or 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 overnight incubation at 65°C. After proteinase K and RNase digestion of the reverse-cross-linked sample, DNA was extracted with phenol-chloroform, precipitated with ethanol, then cleaned with the QIAquick PCR Purification Kit (Qiagen) according to manufacturer instructions.
ChIP libraries were prepared using the KAPA Library Preparation Kit (KAPA Biosystems) per the manufacturer's protocol. Briefly, ChIP DNA was end repaired, followed by addition of an adenine base at the 3' and NEXTflex ChIP-Seq Barcodes (BiooScientific) adaptor ligation. The modified DNA was then size selected on a 2% agarose and purified by using a gel extraction kit (Qiagen). One microliter of size-selected adaptor ligated DNA was initially analyzed by SYBR Green Q-PCR to determine the number of cycles required to reach 50% of complete amplification. Adaptor ligated DNA was PCR amplified with one initial heating step of 98°C for 45 second, followed by the pre-determined number of cycles with a melting temperature of 98°C for 15 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 1 min was performed. Amplified ChIP DNA was subjected to deep sequencing using standard Yale Center for Genome analysis protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Casava 1.8.2 software was used for basecalling.
FASTQ format sequencing reads were mapped to the human genome (hg19 NCBI Build 37) using the BWA version 0.6.1 alignment program with default parameters.
The Model-based Alignment of ChIP-Seq (MACS) program version 1.4.0rc2 was used to identify CTCF and SA1 peaks using default parameters.
The SICER program version 1.1 was used to identify broad regions bound by H3K27me3
Genome_build: hg19
Supplementary_files_format_and_content: The CTCF and SA1 macs1.4 peaks bed output files have a fifth field that is -10*log10(pvalue). The H3K27me3 files have additional SICER fields ChIP_island_read_count, CONTROL_island_read_count, p_value, fold_change, FDR_threshold
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| Submission date |
May 02, 2016 |
| 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 |
GPL11154 |
| Series (2) |
| GSE67783 |
CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells [ChIP-Seq] |
| GSE67893 |
CTCF and CohesinSA-1 Mark Active Promoters and Boundaries of Repressive Chromatin Domains in Primary Human Erythroid Cells |
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| Relations |
| BioSample |
SAMN04931797 |
| SRA |
SRX1738529 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2140683_HSPC_CTCF_2_peaks.bed.gz |
2.3 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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