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| Status |
Public on Mar 05, 2015 |
| Title |
GRO-seq_E2_40m_rep1 |
| Sample type |
SRA |
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| Source name |
GRO-seq_E2_40m
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: ER Positive Breast Cancer Cells
treated with: 100 nM 17β-estradiol (E2) for 40min
molecule subtype: nascent RNA
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| Treatment protocol |
Prior to all treatments, the cells were grown for 3 days in phenol red-free MEM Eagle medium supplemented with 5% charcoal-dextran-treated calf serum. All treatments were performed for 40 min with 100 nM 17β-estradiol, 25 ng/mL TNFα, or both.
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| Growth protocol |
MCF-7 breast cancer cells were obtained from the ATCC and maintained in Minimum Essential Medium Eagle supplemented with 5% calf serum.
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| Extracted molecule |
total RNA |
| Extraction protocol |
MCF-7 cells were seeded at 2.5 million cells per 15 cm dish and treated with Vehicle, E2, TNFα, or E2 + TNFα, as described above. The cells were washed three times with ice-cold PBS, swollen in hypotonic buffer, and collected in ice-cold lysis buffer [10 mM Tris•HCl, pH 7.4, 0.5% NP-40, 10% Glycerol, 3 mM CaCl2, 2 mM MgCl2, 1 mM DTT, 1X protease inhibitor cocktail (Sigma-Aldrich), and SUPERase-In (Ambion)] at 1000 x g for 10 min at 4°C. The cells were then resuspended in 1.5 ml of lysis buffer and pipetted up and down through a narrow opening 30 to 50 times to release the nuclei. The nuclei were pelleted again by centrifugation and washed once with 1 mL of lysis buffer. The nuclear pellets were resuspended in 500 μL of freezing buffer (50 mM Tris•HCl, pH 8.3, 40% glycerol, 5 mM MgCl2, 0.1 mM EDTA, and 4 units of SUPERase-In per mL), counted, and stored in 100 μl aliquots containing 5 x 106 nuclei.
Nuclear run-on and library preparation was performed as previously described (Danko et al., 2013; Hah et al., 2011), with modifications. Briefly, cells were treated for 40 min with 100 nM E2, 25 ng/ml TNFα, or both E2 + TNFα and libraries were prepared from two biological replicates using a circularized ligation-based protocol for adaptor addition used to improve the efficiency of library preparation, reduce sequence bias, and allow for barcoding. The libraries were amplified with indexed primers containing barcodes according to the Illumina TrueSeq small-RNA library prep kit, then sequenced using an Illumina HiSeq 2000. A more detailed protocol is available from the corresponding author (W.L.K.).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
GRO-seq in MCF-7 cells treated for 40 minutes with E2 prior to nuclear run-on reaction
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| Data processing |
library strategy: GRO-seq
Alignment: Short-reads were aligned to the human reference genome (hg19), including autosomes, X-chromosome, and one complete copy of an rDNA repeat (GenBank ID: U13369.1). The BWA software package (Li et al., 2009) was used to align reads and the output was processed using custom Perl scripts, and imported into R for most the analysis.
Genome_build: hg19
Supplementary_files_format_and_content: bed
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| Submission date |
Jul 17, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
W. Lee Kraus |
| E-mail(s) |
lee.kraus@utsouthwestern.edu
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| Organization name |
UT Southwestern Medical Center
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-8511 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (2) |
| GSE59531 |
TNFα Signaling Exposes Latent Estrogen Receptor Binding Sites in Breast Cancer Cells [GRO-seq] |
| GSE59532 |
TNFα Signaling Exposes Latent Estrogen Receptor Binding Sites in Breast Cancer Cells |
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| Relations |
| BioSample |
SAMN02923939 |
| SRA |
SRX656237 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1438937_GRO-seq_E2_40m_rep1.bed.gz |
105.9 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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