|
| Status |
Public on May 18, 2020 |
| Title |
MCF7_shCtrl_noDox_GROseq |
| Sample type |
SRA |
| |
|
| Source name |
breast cancer cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
growth protocol: control (parental cell)
treatment: Control shRNA (scramble) for MCF7 parental cells with DOX-inducible expression of JUN-without DOX treatment
|
| Treatment protocol |
For knockdown of GATA3 or JUN, MCF7 parental or TamR cells were infected with shRNA lentiviruses and selected by puromycin (1 μg/ml) for 3 days to establish GATA3 knockdown MCF7 parental or JUN knockdown TamR stable cell lines. For the Dox-inducible GATA3, JUN-overexpressing cell lines, GATA3 and 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 GATA3 or 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 MCF7 parental cells at the same time, Dox-inducible JUN-overexpressing MCF7 parental 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 |
MCF7 parental cells were maintained in RPMI/1640 supplemented with 10% heat-inactivated FBS (Sigma) and 1% penicillin-streptomycin (P/S). The TamR cells were kept in phenol-red free RPMI/1640 medium supplemented with 10% heat-inactivated charcoal-stripped-FBS and 1% P/S with the addition of 100 nM 4-hydroxytamoxifen (4-OHT, H7904, Sigma). 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 |
Mar 18, 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 |
SAMN11159548 |
| SRA |
SRX5535332 |
