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| Status |
Public on May 22, 2024 |
| Title |
DRIP_seq_shTOP1_pDOX_Rnase_H_OE |
| Sample type |
SRA |
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| Source name |
MCF-7
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: luminal breast cancer
treatment: shTOP1_pDOX_Rnase_H_OE
treatment with dox: WITH DOX
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| Treatment protocol |
The knockdown in TOP1cc ChIP-seq and DRIP-seq experiments were done using the doxycycline inducible shTOP1 plasmid that was kindly obtained from Yilun Liu. For RNase H1 overexpression, RNase H1 plasmid and its empty control plasmid containing only GFP, a kind gift from Dr. Sara Selig (Molecular Medicine Laboratory, Rambam Health Care Campus and Rappaport Faculty of Medicine, Technion, Haifa 31096, Israel), were used. GFP expression was used as an indicative of RNase H1 overexpression. The plasmid was prepared as described in (Sagie et al., 2017).
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| Growth protocol |
MCF7 (HTB-22) were grown in RPMI supplemented with 10% (vol/vol) FBS (GIBCO), glutamine, and penicillin/streptomycin (Beit-Haemek). HMLE cells were grown in Promocell mammary epithelial cell basal media (C-21010) with added supplements (c-93110) whereas MCF10A were grown on DMEM/F12 supplemented with 5% Horse serum, 20 ng/ml EGF, 0.5 mg/ml Hydrocortisone, 100 ng/ml Cholera toxin, 10 mg/ml Insulin and Pen/Strep. MCF-7 cells were synchronized in the G1 cell cycle stage by culturing in serum-free media for 72 hours followed by replacing the media with 10% FBS media for 5 hours. Cells were grown at 37°C under a humidified atmosphere with 5% CO2. Cells were routinely authenticated by STR profiling, tested for mycoplasma, and cell aliquots from early passages were used.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were crosslinked with 1% formaldehyde and quenched with glycine. Fixed cells were washed in lysis buffer. Cells were sonicated using bioruptor sonicator to produce chromatin fragments of ~200-300 bp. The chromatin was immunoprecipitated by incubation with 5 μl of anti-TOP1cc antibody (MABE1048). Immune complexes were captured with protein G Dynabeads. DNA was precipitated by phenol/chloroform/isoamylalcohol extraction. DRIP-seq was done according to the published protocol (Sanz and Chédin, 2019).
library was prepaired using kappa hyperprep kit
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
FASTQ files were trimmed using trim_galore utility. During this step, adapters and low quality bases were filtered out. The fastqc utility was applied to examine data quality.
Trimmed FASTQ files were mapped to the genome, assembly hg38, with hisat2 utility.
Coverage bigwig files were created with deepTools utility bamCompare using ration operation relative to input.
Downstream analysis was performed with R custom scripts.
Assembly: hg38
Supplementary files format and content: bigWig
Library strategy: DRIP-seq
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| Submission date |
Aug 21, 2023 |
| Last update date |
May 22, 2024 |
| Contact name |
Rami Aqeilan |
| E-mail(s) |
ramiaq@mail.huji.ac.il
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| Organization name |
Hebrew University of Jerusalem
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| Street address |
Ein Kerem
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| City |
Jerusalem |
| ZIP/Postal code |
91120 |
| Country |
Israel |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE241307 |
Mapping of TOP1cc and R-loops im MCF-7 after TOP1 KD and/or RNase H OE [CHiP_DRIP_Seq] |
| GSE241309 |
R-loops and Topoisomerase 1 facilitate formation of transcriptional DSBs at gene bodies of hypertranscribed cancer genes |
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| Relations |
| BioSample |
SAMN37096088 |
| SRA |
SRX21436556 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7720905_DRIP_seq_shTOP1_pDOX_Rnase_H_OE_input.bigWig |
245.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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