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Sample GSM5065647 Query DataSets for GSM5065647
Status Public on Feb 05, 2021
Title Luc KD_GRO-seq_E2 rep2
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: Replicate2
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 rep2
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
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
BioSample SAMN17797945
SRA SRX10030370

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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