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| Status |
Public on Aug 04, 2022 |
| Title |
Top1cc-seq_E2_30min_CPT_10min_MCF7_exp2 |
| Sample type |
SRA |
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| Source name |
Human breast cancers
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
treatment: E2_30min_CPT_10min
ip antibody: TOP1cc (TopoGEN, catalog# TG2017-2, lot# 17AG15)
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| Treatment protocol |
For hormone treatments, cells were incubated at 37°C and 5% CO2 for at least 3 days in phenol red-free DMEM (GIBCO/Invitrogen) supplemented with 5% charcoal dextran-stripped FBS (GIBCO/Invitrogen). 17-β-Estradiol (E2; Steraloids, Inc.) was added to a final concentration of 100 nM. TNFalpha (R&D systems) was added to a final concentration of 20 ng/ml. 5α-dihydrotestosterone (DHT, Sigma) was added to a final concentration of 100 nM. The ethanol (EtOH) vehicle control was 0.05% in all samples.
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| Growth protocol |
MCF7 cells were maintained at 37°C and 5% CO2 in Dulbecco's modified Eagle's medium (DMEM, GIBCO/Invitrogen), supplemented with 10% fetal bovine serum (FBS, GIBCO/Invitrogen). LNCAP cells were maintained at 37°C and 5% CO2 in Advanced RPMI 1640 medium (GIBCO/Invitrogen), supplemented with 10% fetal bovine serum (FBS, GIBCO/Invitrogen).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To perform TOP1cc-seq, approximately 2x10e6 cells were treated with 10 μM CPT (Selleckchem, Cat# S1288) for 10 mins and E2 or DHT, and then washed once with ice-cold 1x PBS with freshly added Proteinase inhibitors. The cells were directly in resuspended in Buffer A (50mM Tris-HCl, pH7.4, 1% SDS, 0.5% Empigen BB, 10 mM EDTA) with freshly added 1mM PMSF and Proteinase inhibitors at room temperature for 5 mins and incubate them in ice. Extracts were then sonicated using the Bioruptor® 300 (Diagenode) for 12 cycles [30 s “ON,” 30 s “OFF”] at high power setting for MCF7 cells, and 6 cycles [30 s “ON,” 30 s “OFF”] at high power setting for LNCAP cells. Cell lysate was centrifuged at 21,000 g for 10 min at 4°C and diluted with 9 times of Buffer B (20mM Tris-HCl, pH7.4, 100mM NaCl, 0.5% TritonX-100, 2mM EDTA) with freshly added Proteinase inhibitors. After pre-clear with 20 μl Protein G Dynabeads (Life Technologies, Cat# 10009D), 7 μg TOP1cc (TopoGEN, TG2017-2) was added and incubated with sample over night at 4°C for on a rotator. Immunoprecipitated complexes were collected using 30 μl Protein G Dynabeads, which have been saturated with PBS/1% BSA over night at 4°C, per reaction. And then the immunoprecipitated complexes were successively washed 2 times with pre-cold Washing Buffer I (10mM Tris-HCl, pH7.4, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 0.1% SDS, 1% TritonX-100, freshly added Proteinase inhibitors), 2 times with pre-cold Washing Buffer II (10mM Tris-HCl, pH7.4, 500mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, 1% TritonX-100, freshly added Proteinase inhibitors), 1 time with pre-cold Washing Buffer III (10 mM Tris-HCl pH 7.4, 250 mM LiCl, 0.5% NP-40, 1mM EDTA, 0.5mM EGTA, 0.1% Na-Deoxycholate, freshly added Proteinase inhibitors) and 1 time with pre-cold TE buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA) with 100 mM NaCl and freshly added Proteinase inhibitors. All washes were performed at RT for 5 min on a rotator. SDS Elution Buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1% SDS) was added 1 μl RNase A (Thermo Scientific™, Cat# EN0531) and incubated at 37°C for 2 hr and then added 10 μl Proteinase K (Invitrogen™, Cat# AM2548) and incubated overnight to digest all the proteins. After overnight digestion, DNA was incubated with 5 times volume of PB buffer for 30 mins and purified using QIAquick PCR Purification Kit (QIAGEN, Cat# 28106) according to the manufacturer’s instructions.
The libraries were constructed following Illumina’s Chip-Seq Sample prep kit.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Top1cc_E2_30min_CPT_10min_MCF7_exp2
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| Data processing |
Library strategy: TOP1cc-seq
Basecalls performed using CASAVA version 1.4
Seqencing reads were aligned to the hg38 genome assembly using Bowtie version 2.0.1.
Genome binding peaks were identified using the ‘findPeaks’ command in HOMER with setting of ‘-style factor’: 200 bp peaks with 3-fold enrichment and 0.01 FDR significance over local tags.
To generate histograms for the average distribution of tag densities, position-corrected, normalized tags in 100 bp windows were tabulated within the indicated distance from specific sites in the genome. Clustering plots for normalized tag densities at each genomic region were generated using HOMER and then clustered using Gene Cluster 3.0.
Genome_build: hg38
Supplementary_files_format_and_content: bigWig
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| Submission date |
Aug 12, 2019 |
| Last update date |
Aug 04, 2022 |
| Contact name |
Yuliang Tan |
| E-mail(s) |
yut020@ucsd.edu
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| Organization name |
University of California, San Diego
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| Department |
School of Medicine
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| Street address |
Room 345, CMM West, 9500 Gilman Drive, Mail Code 0648
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92037-0648 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE135723 |
TOP1cc-seq assay in human cancer cells |
| GSE135808 |
Topoisomerase 1 Nicking-dependent HP1gamma Engagement Mediates Acute Activation of Phase-separated Enhancers |
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| Relations |
| BioSample |
SAMN12561572 |
| SRA |
SRX6701228 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4026952_Top1cc_E2_30min_CPT_10min_MCF7_exp2.ucsc.bigWig |
196.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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