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| Status |
Public on Jan 26, 2021 |
| Title |
MCF10A_WT_CTCF_2 |
| Sample type |
SRA |
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| Source name |
MCF10A_WT_CTCF
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF10A
tissue: mammary gland; breast
cell type: epithelial
genotype/variation: Parental
chip antibody: α-CTCF (Millipore 07-729)
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| Treatment protocol |
The p53 samples were treated with nutlin before the fixation.
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| Growth protocol |
Cells were cultured in DMEM/ Ham's F-12 (Biological Industries) supplemented with 100 ng/ml cholera toxin (Sigma), 20 ng/ml epidermal growth factor (EGF)(corning), 0.01 mg/ml insulin(Sigma), 500 ng/ml hydrocortisone (Sigma), 1% penicillin- streptomycin (Gibco), 5% horse serum (Biological Industries) and kept at 37℃ in a 5% CO2 incubator (Thermo Scientific). Parental and transformed cells were subjected to no more than five passages in culture when used in experiments.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked for 10 min at 37°C in 1% formaldehyde followed by quenching with 125 mM glycine for 10 min. Fixed cells were lysed with SDS Lysis Buffer (1% SDS, 50mM Tris pH 8.1, 10mM EDTA) supplemented with protease inhibitor and sonicated for 560 seconds (ME220 sonicator, Covaris). Cleared chromatin was diluted 1:10 with dilution Buffer (16.7mM Tris-HCl 8.1; 1.2mM EDTA; 167mM NaCl; 1.1% Triton) and incubated over night with 5 ug α-p53 (DO-1, Santa-Cruz) bound to magnetic beads (Dynabeads Protein G). Chromatin was washed with low salt buffer (20mM Tris-HCl 8.1; 2mM EDTA; 150mM NaCl; 1% triton; 0.1% SDS), high salt buffer (20mM Tris-HCl 8.1; 2mM EDTA; 500mM NaCl; 1% triton; 0.1% SDS), LiCl buffer (10mM Tris-HCl 8.1; 1mM EDTA; 1% NP-40; 250mM LiCl) at 4°C, and twice with TE (10mM Tris-HCl 8.1; 1mM EDTA) at room temp. Complexes were eluted with Elution buffer (10mM Tris-HCl 8.1; 1mM EDTA; 200mM NaCl; 1% SDS). Crosslinks were reversed with 1 mg/mL Proteinase K overnight at 65°C.
Purified DNA was used to prepare sequencing libraries using NEBNext UltraII DNA Library Prep Kit (New England Biolabs). Library concentration was measured by DNA High Sensitivity Kit (Invitrogen) on a Qubit fluorometer (Invitrogen). Library quality and fragment sizes were assessed on a Bioanalyzer (Agilent).
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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 |
Alignment was done using Bowtie, allowing only unique reads to be considered.
For detecting regions of local read enrichment (peaks), MACS algorithm was applied with default parameters for ChIP-seq.
Genome_build: hg19
Supplementary_files_format_and_content: narrowPeak file that were generated by MACS
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| Submission date |
Nov 12, 2019 |
| Last update date |
Jan 26, 2021 |
| Contact name |
Ofir Hakim |
| E-mail(s) |
Ofir.Hakim@biu.ac.il
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| Organization name |
Bar-Ilan University
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| Street address |
Bar-Ilan University
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| City |
Ramat Gan |
| ZIP/Postal code |
5290002 |
| Country |
Israel |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE140252 |
ChIP seq of CTCF and p53 in MCF10A before and after H-RAS transformation [ChIP-seq] |
| GSE140254 |
Genomic Retargeting of Tumor Suppressors p53 and CTCF Promotes Oncogenesis |
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| Relations |
| BioSample |
SAMN13264610 |
| SRA |
SRX7130725 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4158370_MCF10A_WT_CTCF_2_peaks.narrowPeak.gz |
458.6 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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