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| Status |
Public on Feb 03, 2014 |
| Title |
TMAR 4-DGE analysis |
| Sample type |
SRA |
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| Source name |
Breast cancer cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: breast cancer cell line MCF-7/TAMR-4
genotype: tamoxifen resistant
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| Growth protocol |
The human breast cancer cell line MCF-7 was originally received from The Breast Cancer Task Force Cell Culture Bank, Mason Research Institute. The MCF-7 cells were gradually adapted to grow in low serum concentration. The tamoxifen-sensitive sub-line MCF-7/S0.5 was used to establish tamoxifen-resistant (TAMR) cell lines by long term treatment with 10-6 M tamoxifen. The 4 TAMR cell lines: MCF-7/TAMR-1 (TAMR-1), MCF-7/TAMR-4 (TAMR-4), MCF-7/TAMR-7 (TAMR-7) and MCF-7/TAMR-8 (TAMR-8) were derived from distinct colonies that emerged in cultures of MCF-7/S0.5 cells treated with tamoxifen. The cells were kept within 10 passages throughout the experiments to reduce a possible variability between experimental results
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| Extracted molecule |
total RNA |
| Extraction protocol |
DNA was isolated from the cell lines using DNeasy® Blood & Tissue Kit (Qiagen) according to manufacturer’s protocol. Genomic DNA was digested with BssHII followed by ligation to biotinylated linkers and fragmented by NlaIII (New England BioLabs) cleavage. Because BssHII only cuts unmethylated regions, binding of DNA fragments to streptavidin-conjugated magnetic beads allow separation of the unmethylated and methylated fragments. Bound DNA was ligated to another linker (N) containing the MmeI restriction enzyme recognition site, and then digested with MmeI (New England Biolabs) that generates short sequence tags (20-21 bp, due to enzyme cut floating). The resulting tags were ligated with another linker P7 and amplified by PCR with primers N and P7 for 18 cycles.
Total RNA was extracted from the cell lines with TRI Reagent (Sigma) according to the manufacturer’s protocol. The integrity of the extracted RNA was checked by agarose gel electrophoresis, and the concentration of RNA was estimated by spectrophotometry. Then, mRNA is separated from total RNA by poly-T coated beads and converted to cDNA. The cDNA is isolated and subjected to NlaIII digestion followed by N-ligation, MmeI digestion P7-ligation and PCR to prepare a DEG library in a way analogous to that in MMSDK.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
TAMR 4 is high-dosage tamoxifen selected resistant sublines
DGE-tag
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| Data processing |
Basecalling was performed in BGI-Shenzhen.
A command line tool FASTX-Toolkit implemented in Perl was used to trim the adaptor sequence. The trimmed tags were subjected to quality filtering, so that only tags with sequencing quality higher than 30 for more than 80% of the nucleotides were used for subsequent analysis. For MMSDK tag mapping, we generated a simulated reference library, i.e., BssHII reference library, by in silico enzyme digestion of the human genome (hg19, UCSC) regardless of the methylation state. This library was used as reference for subsequent mapping of the tags in the MMSDK analysis. In the DGE analysis, refMrna (hg19, UCSC) was used as reference for mapping cDNA tags. Subsequently, the Burrows–Wheeler Aligner (BWA) procedure for aligning the MMSDK and DGE tags to the simulated BssHII reference library and refMrna reference library, respectively, was applied.
Genome_build: UCSC hg19
Supplementary_files_format_and_content: Tab-delimited text files (linked as supplementary files on the Series record) include gene expression values (normalized tag number) and DNA unmethylation values (normalized tag number) for each Sample .
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| Submission date |
Sep 06, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Jian Li |
| E-mail(s) |
jianlij@gmail.com
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| Phone |
45+40608651
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| Organization name |
Aarhus University
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| Department |
Institute of Biomedicine
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| Lab |
10
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| Street address |
The Bartholin Building
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| City |
Aarhus |
| ZIP/Postal code |
DK-8000 |
| Country |
Denmark |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE40665 |
Integrative Analyses of Gene Expression and DNA Methylation Profiles in a Breast Cancer Cell Line Model of Tamoxifen-Resistance Indicate a Crucial Role of Cells with Stem-like Properties |
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| Relations |
| SRA |
SRX183873 |
| BioSample |
SAMN01162382 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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