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Sample GSM5065646 Query DataSets for GSM5065646
Status Public on Feb 05, 2021
Title Luc KD_GRO-seq_E2 rep1
Sample type SRA
 
Source name MCF-7
Organism Homo sapiens
Characteristics cell line: MCF-7
cell type: human Estrogen receptor alpha+ breast cancer cells
genotype: LucKD
treatment: 100 nM E2
biological replicate: Replicate1
Treatment protocol 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 (CDCS). For experiments, cells were treated with 100 nM E2 or vehicle (ethanol) for 40 minutes.
Growth protocol MCF-7 cell lines were obtained from the ATCC used for GRO-seq and proliferation assays described herein. MCF-7 cells were maintained in Minimum Essential Medium Eagle supplemented with 5% calf serum.
Extracted molecule total RNA
Extraction protocol 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. (PMID: 7256882)
GRO-seq libraries were prepared as described previously with some minor modifications (PMID: 24564208).
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina Genome Analyzer
 
Description GROseq libraries under Luc KD E2 rep1
Data processing Library strategy: GRO-Seq
The GRO--seq reads were aligned to the hg19 human reference genome using the Bowtie software package (Langmead et al, 2009) .
Mapped reads were further converted to (1) “bed” files for later Metagene and read-density analyses and (2) “wiggle” files counting reads in non-overlapping 200-bp windows across the genome for presentation as genome browser tracks by using the BEDTools software package (Quinlan et al, 2010).
Genome_build: hg19
Supplementary_files_format_and_content: bigwig
 
Submission date Feb 04, 2021
Last update date Feb 06, 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 SAMN17797946
SRA SRX10030369

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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