 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on May 22, 2024 |
| Title |
sBLISS_TOP1_KD_E2_rep2 |
| Sample type |
SRA |
| |
|
| Source name |
MCF-7
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: luminal breast cancer
treatment: TOP1 siRNA + estradiol
|
| Treatment protocol |
Transient transfection of siTOP1 (Dharmacon; SMARTpool siRNA, L-005278-00-0005) and siSc (Dharmacon; D-00181010) was achieved using lipofectamine (ThermoFisher). Cells were seeded to reach a confluency of 60-70% on the day of transfection. Transfection solution was prepared by mixing 1.5ml serum/antibiotic-free RPMI with 30μL lipofectamine and incubating for 5 minutes, before adding a mixture of 1.5ml RPMI and 0.6nmoles of siRNA followed by 20 minutes incubation at RT. The mixture was then added to the cells in a 10cm plate cultured in antibiotic/serum-free media. The media was replaced by fully supplemented media after 5-6 hours, and cells were incubated in the incubator for 48 hours. 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). For estradiol treated cells, MCF-7 cells were hormonally starved by growing in RPMI media supplemented with 10% charcoal stripped fetal bovine serum for 48 hours. Transcription was induced by replacing the media with RPMI media supplemented with charcoal stripped FBS and 100nM β-Estradiol (Sigma-Aldrich) (pre-dissolved in ethanol) and incubated for 1 hour.
|
| 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.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
sBLISS was performed as described in (Bouwman et al., 2020).
library indexing and amplification was performed using NEBNext® Ultra™ II Q5® Master Mix.
in-suspension break labelling in situ and sequencing (sBLISS)
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
BLISS_gene_count_table.txt
|
| Data processing |
FASTQ files were trimmed using trim_galore utility. During this step, adapters (two runs) 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. Resulting BAM files were then UMI de-duplicated using umi_tools, dedup function.
De-duplicated BAM files were converted to SAM files and transformed to BLISS bedGraph files using a custom Python script.
BedGraph files were converted to bigWig files with bedGraphToBigWig utility.
Assembly: hg38
Supplementary files format and content: Tab-delimited text file consisting of BLISS breaks counts per gene per sample.
Library strategy: BLISS-seq
|
| |
|
| Submission date |
Aug 21, 2023 |
| Last update date |
May 22, 2024 |
| Contact name |
Rami Aqeilan |
| E-mail(s) |
ramiaq@mail.huji.ac.il
|
| Organization name |
Hebrew University of Jerusalem
|
| Street address |
Ein Kerem
|
| City |
Jerusalem |
| ZIP/Postal code |
91120 |
| Country |
Israel |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE241305 |
Mapping of physiological DNA double stranded breaks in normal breast cells and breast cancer cells [BLISS] |
| GSE241309 |
R-loops and Topoisomerase 1 facilitate formation of transcriptional DSBs at gene bodies of hypertranscribed cancer genes |
|
| Relations |
| BioSample |
SAMN37096006 |
| SRA |
SRX21436236 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
|
|
|
|
 |
