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| Status |
Public on Aug 15, 2023 |
| Title |
MCF7 cells, shHot_+E2_FOXA1_ChIP-seq |
| Sample type |
SRA |
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| Source name |
MCF7
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| Organism |
Homo sapiens |
| Characteristics |
cell line: Breast Adenocarcinoma cell line MCF7
treatment: shHOTAIRM1, + estrogen
chip antibody: FOXA1 (ab5089 from Abcam)
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| Treatment protocol |
For hormone (estrogen) depletion, cells were changed to charcoal-stripped hormone-free medium (Gibco, Grand Island, NY) supplemented with 10% charcoal dextran-treated fetal bovine serum (Thermal Scientific Hyclone, Logan, UT) for 72 hours. Estrogen induction was performed using 10 nM of 17β-estradiol (Sigma, St. Louis, MO) for the indicated times. Ethanol was used as a vehicle control.
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| Growth protocol |
MCF7 cells were maintained in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum and 1% antibiotics.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation (ChIP) assays were performed with following modifications to previously described high-throughput ChIP protocol (Blecher-Gonen et al, 2013). Briefly, cells were crosslinked in 1% formaldehyde for 10 min, followed by incubation with glycine to stop crosslinking. Cells were collected and washed with ice cold PBS, incubated with cell lysis buffer (5mM PIPES pH 8.0, 85 mM KCl and 0.5% NP40) with protease inhibitor cocktail (Roche) to isolate nuclei. The nuclei were lysed for 30 min on ice using nuclei lysis buffer (12 mM Tris-HCl pH 8.0, 6 mM EDTA pH 8.0, 0.5% SDS) supplemented with protease inhibitor cocktail. Lysate were fragmented with a Bioruptor (Diagenode) to obtain DNA fragments ranging 200-600 bp. The supernatant was incubated with respective antibodies conjugated with Dynabeads Protein G (Invitrogen) in dilution buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, 140 mM NaCl, 0.1% Na-DOC, 1% Triton X-100) overnight at 4°C. The immunocomplexes were collected using Dynamag, washed twice with low-salt buffer (10 mM Tris-HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-DOC) and high-salt buffer (10 mM Tris-HCl pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% Na-DOC) and then once with LiCl buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP40, 0.5% Na-Deoxycholate), and reverse crosslinked overnight followed by DNA extraction.
ChIP–seq libraries were prepared using NEBNext Ultra II DNA library prep kit.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Basecalls performed by Illumina CASAVA 1.8.2
The raw reads (single-end, 100 bp) were aligned to human reference genome hg19 using bowtie (v1.1.0) allowing up to one mismatch.
Genome_build: hg19
Supplementary_files_format_and_content: FPKMs. Peak calling was performed using MACS (v1.4.2) with the default cutoff of P ≤ 1 × 10-8. Clonal reads were automatically removed by MACS.
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| Submission date |
Aug 16, 2019 |
| Last update date |
Aug 15, 2023 |
| Contact name |
Jiejun Shi |
| E-mail(s) |
jiejuns@uci.edu
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| Organization name |
University of California Irvine
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| Department |
School of Medicine
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| Lab |
Li lab
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| Street address |
5270 California Ave
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| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92617 |
| Country |
USA |
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| Platform ID |
GPL21290 |
| Series (1) |
| GSE135894 |
HOTAIRM1 suppresses Estrogen Receptor activity in breast cancer by restricting chromatin accessibility of pioneer factor FOXA1 |
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| Relations |
| BioSample |
SAMN12587400 |
| SRA |
SRX6726414 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4037348_ShH+E_FOXA1.bw |
403.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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