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Sample GSM2151684 Query DataSets for GSM2151684
Status Public on Jun 09, 2016
Title GRO-seq_LucKD_PJ34_MCF7
Sample type SRA
 
Source name GRO-seq in MCF7 cells with shRNA-mediated knockdown of Luciferase as a control with 1 hour 1 µM PJ34 Treatment
Organism Homo sapiens
Characteristics cell line: MCF-7
cell type: human breast cancer cells
gender: female
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.
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.
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).
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina Genome Analyzer
 
Description nascent RNA
Data processing Library strategy: GRO-Seq
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
 
Submission date May 12, 2016
Last update date Feb 04, 2021
Contact name W. Lee Kraus
E-mail(s) lee.kraus@utsouthwestern.edu
Organization name UT Southwestern Medical Center
Street address 5323 Harry Hines Blvd.
City Dallas
State/province TX
ZIP/Postal code 75390-8511
Country USA
 
Platform ID GPL9052
Series (1)
GSE74142 NAD+ Analog-sensitive PARPs Reveal a Role for PARP-1 in Transcription Elongation
Relations
BioSample SAMN04994153
SRA SRX1758468

Supplementary file Size Download File type/resource
GSM2151684_GRO-seq_LucKD_PJ34_MCF7.bed.gz 136.5 Mb (ftp)(http) BED
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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