|
| Status |
Public on May 06, 2020 |
| Title |
Untreated replicate 1 |
| Sample type |
SRA |
| |
|
| Source name |
Basal breast cancer cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-468 cells
passages: 6 to 14
treatment: untreated
|
| Treatment protocol |
Cells were harvested by trypsinization. After adding media to stop trypsin, cells were centrifuged at 500 x g for 5 min at room temperature.
|
| Growth protocol |
MDA-MB-468 cells were grown to ~ 70% confluence on 140 cm2 dishes and treated with EGF (100 ng mL-1) for 72 h in biological duplicate. Untreated cells were used as an appropriate control. Whole cell extracts (~ 9 x 105 cells) were collected by centrifugation at 500 x g for 5 min at 4ºC and washed twice in ice-cold PBS1x.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
MDA-MB-468 cells were grown to ~ 70% confluence on 140 cm2 dishes and treated with EGF (100 ng mL-1) for 72 h in biological duplicate. Untreated cells were used as an appropriate control. Whole cell extracts (~ 9 x 105 cells) were collected by centrifugation at 500 x g for 5 min at 4ºC and washed twice in ice-cold PBS1x. Total RNA were extracted using RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and subjected to DNase I treatment using the RNase-free DNase set (Qiagen). The total RNA was then analyzed using Agilent RNA 6000 Nano Kit on the Agilent 2100 Bioanalyzer System to determine RNA concentration, qualify RNA Integrity Number (RIN) which was high with RIN = 10 for all samples and confirm removal of DNA contamination. After quality control of RNA, the ERCC RNA Spike-In (Ambion) was added to RNA samples (RNA samples: RNA Spike-In control diluted at 1/100, 5:1).
RNA sequencing libraries were prepared from 1µg of total RNA using the Illumina TruSeq Stranded mRNA Library preparation kit which allows to perform a strand specific sequencing. A first step of polyA selection using magnetic beads is done to focus sequencing on polyadenylated transcripts. After fragmentation, cDNA synthesis was performed and resulting fragments were used for dA-tailing followed by ligation of TruSeq indexed adapters. PCR amplification was finally achieved to generate the final barcoded cDNA libraries. The 4 libraries were equimolarly pooled and subjected to qPCR quantification using the KAPA library quantification kit (Roche). Sequencing was carried out on the NovaSeq 6000 instrument from Illumina based on a 2*100 cycle mode (paired-end reads, 100 bases) using a S1 flow cell in order to obtain around 35 million clusters (70 million raw paired-end reads) per sample
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
tablecounts_raw.csv
D203T02
|
| Data processing |
Raw sequencing reads were aligned on the human hg38 reference genome using the STAR mapper [7] (2.5.3a), up to the generation of a raw count table per gene (Gencode annotation v26). Expressed genes (TPM>=1 in at least one sample) have then been selected for downstream analysis. The raw count table was then normalized using the TMM method from the edgeR R package [5] (v3,22,3), and the limma voom (v3.36.3) functions were applied to detect genes with differential expression between untreated and EGF samples. Genes with an adjusted pvalue < 0,05 and a log2 fold-change > 1 were called significant.
Genome_build: hg38
Supplementary_files_format_and_content: table of raw counts per gene
|
| |
|
| Submission date |
May 05, 2020 |
| Last update date |
May 07, 2020 |
| Contact name |
Nicolas Servant |
| E-mail(s) |
[email protected]
|
| Organization name |
Institut Curie
|
| Street address |
26 rue d'ulm
|
| City |
Paris Cedex 05 |
| ZIP/Postal code |
75248 |
| Country |
France |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE121663 |
Role of iron in the regulation of the epithelial-to-mesenchymal transition (RNA-seq) |
| GSE121664 |
Role of iron in the regulation of the epithelial-to-mesenchymal transition |
|
| Relations |
| BioSample |
SAMN14838364 |
| SRA |
SRX8251859 |
