|Public on May 18, 2020
|breast cancer cells
|cell line: T47D
treatment: GATA3 shRNA (sh9301) for T47D cells with DOX-inducible expression of JUN-with DOX treatment
|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.
|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.
|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.
|Illumina HiSeq 3000
|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.
|Dec 11, 2019
|Last update date
|May 19, 2020
|Zhijie Jason Liu
|Universality of Texas Health Science Center at San Antonio
|Department of Molecular Medicine
|7703 Floyd Curl Drive
|Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance [GRO-Seq]
|Enhancer reprogramming driven by high-order assemblies of transcription factors promotes phenotypic plasticity and breast cancer endocrine resistance