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Sample GSM4214089 Query DataSets for GSM4214089
Status Public on May 18, 2020
Title T47D_both_GROseq
Sample type SRA
 
Source name breast cancer cells
Organism Homo sapiens
Characteristics cell line: T47D
treatment: GATA3 shRNA (sh9301) for T47D cells with DOX-inducible expression of JUN-with DOX treatment
Treatment protocol For knockdown of GATA3, T47D cells were infected with shRNA lentiviruses and selected by puromycin (1 μg/ml) for 3 days to establish GATA3 knockdown T47D stable cell lines. For the Dox-inducible JUN-overexpressing cell lines, JUN cDNAs (Open Biosystems) were cloned into the pInducer 20 destination vector (Meerbrey et al., 2011) using the Gateway system (Invitrogen). Lentivirus was produced in 293T cells and used to infect cells in media containing polybrene (8 μg/ml). Cells were selected for 4 days by 500 μg/mL G418 (Invitrogen) after virus infection to get inducible overexperssion stable lines. To induce JUN protein expression, 1 μg/ml doxycycline was added into culture media for about 48 h before collection for experiments. For knockdown of GATA3 and overexpression of JUN in T47D cells at the same time, Dox-inducible JUN-overexpressing T47D stable cell line was further infected with shGATA3 lentiviruses and selected by puromycin (1 μg/ml) for 24 h, then 1 μg/ml doxycycline was added into culture media to induce JUN overexpression for about 48 h before collection for experiments.
Growth protocol T47D cells were maintained in RPMI/1640 supplemented with 10% heat-inactivated FBS (Sigma) and 1% penicillin-streptomycin (P/S). All cells were kept at 37 °C in a humified incubator with 5% CO2.
Extracted molecule total RNA
Extraction protocol Cells were subjected to nuclear run-on for 5minutes at 30°C with BrU labeling. The run-on RNAs were pull down by BrU beads and subjected to library preparation for deep sequencing.
We followed a previous published protocol (Ingolia et al., 2009 Science; Wang et al., 2011 Nature). Briefly, the run-on RNA were first added with a polyA tracts by RNA polyA polymerase. This polyA tail enables the run-on RNA to be reverse transcribed into single strand cDNA by Reverse Transcriptase. The cDNA was circularized by CircLigase (Epicentre) and re-linearized by APE I, subsequently subjected to PCR amplification and deep sequencing.
 
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 3000
 
Description Nascent RNA
Data processing Library strategy: GRO-seq
Basecalls performed using Illuminal CASAVA software.
GRO-seq reads were aligned to human genome (hg19) using bowtie with “--best --strata –m 1 –v 2” parameters. Duplicated reads were eliminated for subsequent analysis.
To balance the clonal amplification bias and total useful reads, only at most three reads was allowed for each unique genomic position.
edgeR 3.16.5 was used to call the differential eRNAs.
Genome_build: hg19
Supplementary_files_format_and_content: bigWig
 
Submission date Dec 11, 2019
Last update date May 19, 2020
Contact name Zhijie Jason Liu
E-mail(s) liuz7@uthscsa.edu
Organization name Universality of Texas Health Science Center at San Antonio
Department Department of Molecular Medicine
Lab Liu lab
Street address 7703 Floyd Curl Drive
City San Antonio
State/province TEXAS
ZIP/Postal code 78229
Country USA
 
Platform ID GPL21290
Series (2)
GSE128452 Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance [GRO-Seq]
GSE128460 Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance
Relations
BioSample SAMN13541465
SRA SRX7348006

Supplementary file Size Download File type/resource
GSM4214089_T47D_both_minus.bw 71.5 Mb (ftp)(http) BW
GSM4214089_T47D_both_plus.bw 54.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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