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| Status |
Public on Oct 05, 2016 |
| Title |
EPRS shRNA 84 Replicate 3 |
| Sample type |
SRA |
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| Source name |
Tam resistant MCF7
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| Organism |
Homo sapiens |
| Characteristics |
treatment: EPRS shRNA 84
phenotype: Tamoxifen Resistance
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| Treatment protocol |
For siRNA, cells were reverse-transfected with 20nM siRNA using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer’s protocol. Media is changed after overnight incubation. Pre-validated EPRS shRNAs in the pLKO.1 vector were obtained from Sigma. For shRNA lentivirus production, 293T cells were transfected with viral plasmids using Lipofectamine 2000 according to standard protocols. Virus-containing supernatant was collected at 48, 72, 96, and 120 hours after transfection, pooled, and frozen at -80°C to eliminate carry-over 293T cells. For shRNA lentivirus infection of target cells, lentivirus was thawed on ice and concentrated using centrifugal filters (Amicon). ShRNA lentivirus and target cells were simultaneously added to six-well plates, centrifuged at 2250rpm for 30 minutes at room temperature, then incubated at 37°C overnight. Media was changed the next morning and cells were allowed to recover for six to eight hours, after which two μg/mL puromycin was added to select shRNA-expressing cells. Puromycin was reduced to a maintenance dose of one μg/mL after 24-48h. After cells expressing EPRS shRNA vectors being generated, MCF7 TamR cells were infected with control or one of three unique EPRS shRNA sequences.
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| Growth protocol |
MCF7 TamR cells, obtained from the Rachel Schiff Lab (Houston, TX), were cultured in phenol red-free RPMI media (Life Technologies) with 10% charcoal-stripped fetal bovine serum (FBS) (Sigma), one percent penicillin/streptomycin (Life Technologies), and 100nM 4-hydroxytamoxifen (4-OHT). 4-OHT was withdrawn for functional assays.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Seventy-two hours after infection, cells were lysed and total RNA was purified using a Qiagen RNeasy kit according to the manufacturer’s protocol.
RNA was stored at -80°C until submission to the Mount Sinai Genomics Core Facility for ribosomal RNA depletion, cDNA library preparation, and sequencing using paired-end, 100nt reads on an Illumina HiSeq 2000.
Ribosomal RNA depletion
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Sample 12
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| Data processing |
The pair-ended sequencing reads were aligned to human genome hg38 using star aligner (version 2.5.0b).
Following read alignment, featureCounts was used to quantify gene expression at the gene level based on GENCODE gene model release 22.
Read counts were further processed and normalized using packages edgeR and limma to obtain normalized and log2 transformed expression levels.
Genome_build: hg38
Supplementary_files_format_and_content: Read counts in tab delimited
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| Submission date |
May 05, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
XIANXIAO ZHOU |
| E-mail(s) |
xianxiao.zhou@mssm.edu
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| Phone |
2128248964
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| Organization name |
Ichan School of Medicine at Mount Sinai
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| Department |
Department of Genetics and Genomic Sciences
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| Lab |
The Multiscale Network Modeling Laboratory
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| Street address |
1470 Madison AVE
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE81162 |
EPRS is a Critical Regulator of Cell Proliferation and Estrogen Signaling in ER+ Breast Cancer |
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| Relations |
| BioSample |
SAMN04870089 |
| SRA |
SRX1714559 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2144029_EPRS_shRNA_84_Replicate_3.txt.gz |
260.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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