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| Status |
Public on Oct 22, 2016 |
| Title |
TMR |
| Sample type |
SRA |
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| Source name |
TMR cells cultured in medium with vehicle control.
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| Organism |
Homo sapiens |
| Characteristics |
cell line: TamR subline
cell line description: Tamoxifen-resistant cell line
agent: vehicle control
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| Treatment protocol |
MCF7 and TMR cells were seeded into 6-well tissue culture plates. The following day, media was replaced by phenol red-free DMEM medium supplemented with 10% charcoal/dextran-treated fetal bovine serum and the cells were allowed to grow for two days. then, cells were treated with 4-hydroxytamoxifen or vehicle for another 24 hours.
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| Growth protocol |
MCF-7 cells (ATCC HTB-22) were cultured in complete medium (DMEM, 10% FBS, Pen-Strep). The tamoxifen resistant (TMR) sublines were generated by maintaining the parental MCF-7 cells in phenol red-free medium supplemented with 10% charcoal/dextran stripped fetal bovine serum containing 1 µM 4-hydroxytamoxifen (Sigma) for over 8 months. The cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 (v/v).
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the TRIzol Reagent (Invitrogen) from MCF-7 parental and TMR cells treated with or without 4-hydroxytamoxifen for 24 hours.
RNA-Sequencing (RNA-Seq) libraries were prepared from 1 μg total RNA using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA) according to the manufacturer’s protocol. Briefly, poly-adenylated mRNA was purified from total RNA and rRNA removed by two rounds of binding to magnetic poly-T beads. This was followed by RNA fragmentation by incubation in the presence of divalent cations at 94ºC for 5 minutes. Double-stranded cDNA was generated by random-primed first-strand synthesis with SuperScript II reverse transcriptase and subsequent second strand synthesis with RNase H and DNA Polymerase I. The cDNA was then blunt-ended with T4 and Klenow DNA polymerases to remove 3′-overhangs and fill in 5′-overhangs, phosphorylated with T4 PNK, and then 3′-A tailed by incubation with Klenow Fragment (3´→5´ exo–) and dATP. Illumina paired-end (PE) adapters were ligated, followed by purification wit AMPure XP beads. The library was enriched by high-fidelity PCR amplification (15 cycles) with Phusion DNA Polymerase (Finnzymes Oy) and adapter-specific primers.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Illimuna next-generation sequencing (2x100bp, paired-end)
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| Data processing |
Image processing, base calling, quality scoring (Phred), FASTQ file generation, and sample demultiplexing were executed by HiSeq Control Software with Real Time Analysis (HCS 1.5/RTA 1.13) and CASAVA 1.8 software (Illumina; San Diego, CA).
The FASTQ-formatted sequence data was analyzed using a standard TopHat-Cufflinks workflow (Trapnell et al., 2012). In brief, sequence reads were mapped to the reference human genome assembly (Feb. 2009, GRCh37/hg19) with TopHat software (Trapnell et al., 2009). Subsequently, the Cufflinks package (Trapnell et al., 2010) was applied for transcript assembly, quantification of normalized gene and isoform expression in FPKM (fragments per kilobase of transcript per million mapped fragments), and testing for differential expression (Cuffdiff).
Processed data are Cufflinks output files (tracking) and contain Gene IDs, locus, and FPKM values.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include Gene ID, locus, and FPKM values for each gene and were prepared from the Cufflinks output files (tracking).
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| Submission date |
Oct 22, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Clifford G. Tepper |
| E-mail(s) |
cgtepper@ucdavis.edu
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| Phone |
916-734-7195
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| Organization name |
UC Davis School of Medicine
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| Department |
Biochemistry and Molecular Medicine
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| Street address |
4645 2nd Avenue, Room 2300A
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| City |
Sacramento |
| State/province |
CA |
| ZIP/Postal code |
95817 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE62613 |
Nuclear TIGAR mediates an epigenetic-metabolic loop via Nrf2 for cancer therapeutics resistance |
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| Relations |
| BioSample |
SAMN03135263 |
| SRA |
SRX738350 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1530031_TMR_genes_fpkm.txt.gz |
807.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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