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| Status |
Public on Jun 09, 2016 |
| Title |
RNA-seq_LucKD_DRB_MCF7_1 |
| Sample type |
SRA |
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| Source name |
RNA-seq in MCF7 cells with shRNA-mediated knockdown of Luciferase as a control with 3 hours 100 µM DRB treatment
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: human breast cancer cells
gender: female
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| Treatment protocol |
GRO-seq: Prior to all experiments, MCF-7 cells were grown for 3 days in phenol red-free MEM Eagle medium supplemented with 5% charcoal-dextran-treated calf serum. RNA-seq: Prior to all experiments, MCF-7 cells were grown for 3 days in phenol red-free MEM Eagle medium supplemented with 5% charcoal-dextran-treated calf serum and 1 µg/mL Doxycycline for 4 days prior to treatment with DRB or vehicle (DMSO) treatment for 3 hours prior to RNA harvest.
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| Growth protocol |
MCF-7 cells were maintained in Minimum Essential Medium Eagle supplemented with 5% calf serum containing 0.5 μg/mL puromycin and 800 μg/mL G418.
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| Extracted molecule |
total RNA |
| Extraction protocol |
GRO-seq: MCF-7 cells were washed three times with ice-cold PBS and then resuspended for swelling in ice-cold Hypotonic Lysis Buffer [10 mM Tris•HCl, pH 7.4, 0.5% NP-40, 10% Glycerol, 3 mM CaCl2, 2 mM MgCl2, and 1 mM DTT containing 1x protease inhibitor cocktail (Sigma-Aldrich) and 4 units/mL SUPERase-In (Ambion)]. The swollen cells were collected by centrifugation at 1000 RCF for 10 min at 4°C and then resuspended in 1.5 ml of lysis buffer and pipetted up and down through a narrow opening tip 30 to 50 times to lyse the cells and release the nuclei. The nuclei were collected by centrifugation and washed once with 1 mL of Hypotonic Lysis Buffer. After a final collection by centrifugation, the resulting pellets of nuclei 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/mL of SUPERase-In per mL), counted, frozen in liquid nitrogen in 100 μL aliquots containing 5 x 106 nuclei, and stored at -80°C until use. RNA-seq: Following 3 hours of treatment, RNA was harvested using QIAGEN RNeasy Kit.
GRO-seq: Nuclear run-on and GRO-seq library preparation were performed as previously described {Core, 2008;Hah, 2011}. Briefly, nuclear run-on reactions were performed for ~100 bases in the presence of sarksoyl (to prevent reengagement of RNA polymerases), rNTPs, α32P-CTP, and 5-bromo-UTP. The nascent RNAs were isolated, hydrolyzed to ~100 bases, and enriched using α-BrdUTP antibody-conjugated agarose beads (Santa Cruz). The bound RNAs were washed several times and eluted. The 5’ RNA cap was removed and the ends were repaired in preparation for adapter ligation. Small RNA adapters were ligated to the 5’ end, followed by another bead binding enrichment using α-BrdUTP antibody-conjugated agarose beads. These steps were repeated using a 3’ adapter. The resulting RNAs were reverse transcribed, amplified using PCR, and analyzed by high throughput sequencing using an Illumina 1G Genome Analyzer. RNA-seq: RNA was prepared for sequencing as previously described (Sun, M. et al. Mol. Cell 2015).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
oligo(dT)-purified RNA
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| Data processing |
Trimmed human GRO-seq reads were aligned to the human reference genome (hg19) using the bwa aligner {Li and Durbin, 2009} with default settings (uniquely aligned, 2 mismatches allowed, and 19 bp seed sequence). The 5’-most base pair from each read was used in all analyses, with no more than 2 duplicates allowed at any genomic location. RNA-seq reads were mapped to the hg19 human genome using Tophat and converted to bed files.
Genome_build: hg19
Supplementary_files_format_and_content: bed
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| Submission date |
May 12, 2016 |
| 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 |
GPL18573 |
| Series (1) |
| GSE74142 |
NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation |
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| Relations |
| BioSample |
SAMN04994156 |
| SRA |
SRX1758471 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2151687_RNA-seq_LucKD_DRB_MCF7_1.bed.gz |
449.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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