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| Status |
Public on Sep 15, 2023 |
| Title |
INPUT_CTE_1616 |
| Sample type |
SRA |
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| Source name |
MCF7
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
cell type: MCF7 Breast cancer
chip antibody: NA
agent: E2
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| Treatment protocol |
E2 (10 nM, Sigma-Aldrich) or ethanol (vehicle) was added 1h prior to fixation
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| Growth protocol |
Four 15 cm plates of control and MAF-overexpressing MCF7 cells per condition were prepared at 70-80% confluency. Cells were cultured in hormone-deprived medium [phenol-red free DMEM (Gibco) supplemented with 5% charcoal-stripped FBS (Capricorn Scientific), glutamine 0.29 mg/mL, penicillin 100 units/mL and streptomycin 0.1 mg/mL for 72 h before E2 (10 nM, Sigma-Aldrich) or ethanol (vehicle) addition.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked in 1% formaldehyde in DMEM for 10 min at RT. Next, glycine was added to a final concentration of 0.125 M and cells were incubated for 5 min at RT to stop the fixation. After two washes with ice-cold PBS, cells were harvested by gently scrapping on ice and centrifuged at 3,000 x g for 5 min. Pellets were stored at 80ºC until use. Chromatin preparation: cells were lysed in hypotonic lysis buffer (5mM Pipes pH 8; 85mM KCl; 0.5% NP40; plus protease inhibitors). Nuclei were recovered by centrifugation, lysed in nuclear lysis buffer (50mM Tris HCl pH 8; 1% SDS; 10mM EDTA plus protease inhibitors) and sonicated with a Bioruptor Pico, Diagenode (20 cycles 30” ON, 30” OFF) to yield chromatin fragments of 150-300 bp. For ChIP experiments, chromatin (25 µg DNA) was diluted 1/10 in ChIP Buffer (16.7mM Tris HCl pH 8; 167mM NaCl; 1.2mM EDTA; 1.1% Triton x100 plus protease inhibitors) and mix with 2% Drosophila Spike-in chromatin. Aliquots were removed as input material (1%). ChIP samples were incubated with the specific antibody (5 µg) plus Spike-in antibody (1µg) overnight 4ºC, and immunoprecipitated with Protein A agarose beads (Diagenode) during 2 h at 4°C. Beads were washed 3 times with low-salt buffer (20mM Tris HCl pH8; 150 mM NaCl; 2mM EDTA; 0.1% SDS; 1% Triton X-100) and once with high-salt buffer (20mM Tris HCl pH8; 400 mM NaCl; 2mM EDTA; 1% Triton X-100; 0.1% SDS). ChIPed material was eluted from the beads in Elution buffer (1% SDS, 100 mM NaHCO3) at 65°C in a shaker (1000 rpm) for 1 h. Eluted material and inputs were de-crosslinked overnight at 65°C, treated with Proteinase K and the DNA purified using the QIAquick PCR purification kit (Qiagen). The following antibodies were used in the ChIP experiments: H3K4me3 (Diagenode, C15410003); H3K27Ac (Millipore, #07–360); Drosophila H2Av (Active Motif #61686).
Libraries were prepared according to Illumina instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 2000 |
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| Description |
Input in E2 stimulated MCF7 cells
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| Data processing |
Sequence reads of human with fly spike-in samples were mapped to the human + fruit fly genome (hg19+dm3) using BOWTIE (PubMed ID 19261174) with the option -m 1.
Peak detection was performed with MACS (PubMed ID 18798982)
Assembly: hg19/dm3
Supplementary files format and content: Tab delimeted files with macs peaks
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| Submission date |
May 17, 2023 |
| Last update date |
Sep 15, 2023 |
| Contact name |
Adria Caballe |
| E-mail(s) |
adria.caballe@irbbarcelona.org
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| Organization name |
IRB
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| Department |
biostatistics
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| Street address |
C/ Baldiri Reixac 10
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| City |
Barcelona |
| ZIP/Postal code |
08028 |
| Country |
Spain |
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| Platform ID |
GPL30173 |
| Series (2) |
| GSE232389 |
MAF Amplification licenses Estrogen Receptor α to Drive Breast Cancer Metastasis. |
| GSE232706 |
MAF Amplification licenses Estrogen Receptor α to Drive Breast Cancer Metastasis.[ChIP-Seq] |
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| Relations |
| BioSample |
SAMN35133002 |
| SRA |
SRX20401569 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not applicable for this record |
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