|
| Status |
Public on Aug 18, 2020 |
| Title |
Human 436_20B_PAC-3 |
| Sample type |
SRA |
| |
|
| Source name |
MDA-MB-436
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: TNBC
paclitaxel sensitivity: Taxol-resistant
replicate: GD_RNA_14 and 4
agent: 20nM Paclitaxel
|
| Treatment protocol |
Taxol-resistant cells were grown in presence of 20nM of Paclitaxel at all time. Parental cells (MDA-MB-436 and Hs 578T) were grown in DMEM with 1:1000 DMSO at all time.
|
| Growth protocol |
MDA-MB-436 and HS 578T cells were grown in DMSO supplemented with 10%FBS and Penicilin/Streptomycin (100 U/mL Penicilium and 100 μg/mL. Streptomycin). Taxane-resistant cells were generated upon long-term exposure to increasing concentrations of paclitaxel at a starting concentration 0.05 nM with increments (dose and time adjusted to the growth and survival of adapting cells), until the cytotoxic concentration of 10 to 20 nM was reached with the resistant cells growing at a similar rate than the parental cells (>6 months).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the qiaquick purification kit according to manufacturer's protocol
RNA samples were quantified by qubit (Life Technologies) and quality by Agilent Bioananlyzer. All samples had RIN score >8 were library prepared using TruSeq Stranded mRNA kit (Illumina). Two hundred nanograms total RNa from 30 RNA samples were purified for polyA tail containing mRNA molecules using poly-T oligo attached magnetic beads , following purification the RNA was fragmented. The cleaved RNA fragments were copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using RNase H and DNA Polymerase I. A single “A” based were added and adapter ligated followed by purification and enrichment with PCR to create cDNA libraries.
Final cDNA libraries were size validated using Agilent Bioanalyzer and concentration validated by qPCR (Kapa Biosystems/Roche). All libraries were normalized to 10nM and pooled together, denatured with 0.2N NaOH and diluted to a final concentration of 8 pM. Pooled libraries were loaded onto cBot (Illumina) for cluster generation. Clustered flow cell was then sequenced Paired-end 100 cycles V3 using Illumina Hiseq2000 to achieve ~30 million reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
GD_RNA_24
|
| Data processing |
Reads were aligned to hg19 using tophat (2.0.8) with default parameters
Transcripts were merged using cufflinks (2.1.1) and differential expression computed for all relevant contrasts with default parameters
Genome_build: hg19
Supplementary_files_format_and_content: csv file showing mean FPKM value for all identified transcripts
|
| |
|
| Submission date |
Apr 25, 2018 |
| Last update date |
Aug 18, 2020 |
| Contact name |
Genevieve Deblois |
| E-mail(s) |
[email protected]
|
| Organization name |
University Health Network, University of Toronto
|
| Department |
Medical Biophysics
|
| Lab |
Mathieu Lupien
|
| Street address |
101 College street
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G 1L7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE113685 |
Metabolic adaptations underlie epigenetic vulnerabilities in chemoresistant breast cancer. [RNA-Seq2] |
| GSE113687 |
Metabolic adaptations underlie epigenetic vulnerabilities in chemoresistant breast cancer |
|
| Relations |
| BioSample |
SAMN08982269 |
| SRA |
SRX3995797 |
