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| Status |
Public on Mar 01, 2014 |
| Title |
HD3 input control [ChIP-Seq] |
| Sample type |
SRA |
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| Source name |
erythroblast cell line
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| Organism |
Gallus gallus |
| Characteristics |
cell line: HD3 erythroblast cell line
cell line details: clone A6 of the line LSCC
chip antibody: none
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| Treatment protocol |
To induce erythroid differentiation, HD3 cells at a density of 8*10^5 cells/ml were incubated in the above medium additionally containing 10mM HEPES (pH 8.0) and 20 mM iso-H-7 (1-(5-Isoquinolinylsulfonyl)-3-methylpiperazine dihydrochloride, Fluka, Seelze, Germany) at 428C in 100% air atmosphere (Nicolas,R.H, 1991). To estimate the percentage of dead cells, seven parts of the cell suspension in growth medium were mixed with one part of 0.4% trypan-blue solution (Gibco, Carlsbad, CA, USA) and the percentage of stained blue cells was determined under a light microscope. To estimate the percentage of cells containing hemoglobin, 25 ml of 0.4% (w/v) solution of benzidine (Sigma, St.Louis, MO, USA) in 4% (v/v) acetic acid was mixed with 1 ml of 30% H2O2 and then 25 ml of the cell suspension in growth medium was added. After 10 min staining, the percentage of stained dark blue cells was determined under a light microscope (Rowley,P.T.,1985).
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| Growth protocol |
The avian erythroblastosis virus-transformed chicken erythroblast cell line HD3 (clone A6 of the line LSCC (Beug, 1979;Beug, 1979) and the DT40 lymphoid cell line (CRL-2111, ATCC) were grown in Dulbecco’s Modified Eagle Medium supplemented with 2% chicken serum and 8% fetal bovine serum at 37ºC with 5% CO2. In the case of DT40 cells, the medium additionally contained 50 μM β-mercaptoethanol.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation was performed essentially as described prevoiusly (Orlando, 2000) with modifications presented in the Life Technologies protocol (http://tools.lifetechnologies.com/content/sfs/manuals/DynabeadsProteinG_man.pdf). About 108 logarithmically growing cells were fixed with 1% v/v formaldehyde in DMEM/F-12 (1:1) medium (Invitrogen, 42400-010) at room temperature for 8 min. The fixed cells were pelleted at 700xg for 4 min at 4oC, washed with PBS containing 1 mM AEBSF and 2 ul/ml of a protease inhibitor cocktail (Sigma, P8340), pelleted again and resuspended in 500 μl of lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA). The lysate was incubated for 10 min on ice, and sonicated with a Cole-Parmer CP750 ultrasonic processor (30% amplitude, 40 cycles for 3 s each with 10 s intervals). Cell debris was removed in a microcentrifuge (10 min, 13000 rpm, 4°C), and the supernatant was diluted 10-fold with 16.7 mM Tris-HCl, pH 8.0, 16.7 mM NaCl, 1.2 mM EDTA, 1% Triton X-100, 0.01% SDS, 1 mM AEBSF, and 1 μl/ml of the protease inhibitor cocktail. Aliquots were taken at this stage for use as an input control. The cell lysates were precleared by incubation with the Dynabeads Protein G magnetic beads (Life Technologies) and then incubated with 30 μg of anti-CTCF antibodies overnight at 4°C with rotation. Rabbit custom anti-CTCF antibodies were prepared against the part of the chicken CTCF gene encoding the N-terminal region of the protein comprising amino acids 86-233. After incubation with antibodies, DNA-protein complexes were collected with the Dynabeads Protein G magnetic beads and washed as described by the manufacturer (Life Technologies) and eluted by two incubations for 15 min in elution buffer (1% SDS, 0.1 M NaHCO3) at room temperature.
The part of the chicken CTCF gene encoding the N-terminal region of the protein comprising amino acids 86–233 was cloned in the pET-28a(+) vector (Novagen). Protein expression was induced with IPTG and purification was carried out through a Ni(+) charged column. Once the protein was obtained, antibodies were generated by immunizing male New Zealand white rabbits. Antibody titers were estimated by ELISA.
DNA samples were purified using the QIAquick Gel Extraction Kit as recommended by the manufacturer (Qiagen).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
HD3 input
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| Data processing |
Basecalling with CASAVA 1.8.2 software
Mapping to galGal4 genome with bowtie2 aligner
ChIP peak calling with MACS software with P-value threshold of 10^-5. One sample was used as input DNA control.
Genome_build: Gallus_gallus-4.0
Supplementary_files_format_and_content: Bed file reports MACS peaks.
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| Submission date |
Oct 29, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Artem V Artemov |
| Organization name |
Medical University of Vienna
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| Department |
Center for Brain Research
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| Street address |
Spitalgasse 4
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL16133 |
| Series (2) |
| GSE51846 |
Clustering of CpG islands constitutes an important determinant of the interphase chromosome 3D organization [ChIP-Seq] |
| GSE51939 |
Clustering of CpG islands constitutes an important determinant of the interphase chromosome 3D organization |
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| Relations |
| BioSample |
SAMN02387920 |
| SRA |
SRX370119 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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