|
| Status |
Public on May 27, 2014 |
| Title |
NS2_RNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
MCF7_unstimulated
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
cell type: breast cancer cells
stimulated with: none (unstimluated (NS) control)
|
| Treatment protocol |
The next day, cells were either stimulated with 5μM Nutlin-3a (in case of p53-WT cells) or left untreated.
|
| Growth protocol |
Wild-Type (WT) MCF-7 cells were plated onto 24-well plates (60000 cells/well) in RPMI medium (+ L-glutamaat, Gibco) supplemented with 10% fetal bovine serum, 0.4mM sodium pyruvate (Gibco), 100μm/ml penicillin/streptomycin (Invitrogen), 1x non-essential aminoacids (Gibco) and 10μg/ml Insulin (Sigma).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After 24h, cells were washed in PBS (Gibco) and prepared for RNA extraction according to the RNeasy protocol (Qiagen), yielding around 2 μg of total RNA per sample.
Illumina TruSeqTM RNA Sample preparation guide using the appropriate indices
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Non stimulated RNA-seq replicate 2 (NS2)
|
| Data processing |
An initial filtering was performed to remove reads containing residual adapter sequences (fastx_clipper, version 0.013, option –M15).
Next, a quality report was generated using the FastQC software (version 0.9) checking the reads for PHRED quality (> 20), read length (> 20) and primer- contamination.
Filtered reads were mapped to the human reference genome hg19 (UCSC) using TopHat (v1.3.3) with its default settings.
Gene expression was measured with HT-Seq (–str=no parameter, version 0.5.3p3) using the human RefSeq annotation, release 42.
DESeq was used to calculate differential expression between Nutlin3a-stimulated and non-stimulated samples. Only genes containing more then 1 read per million in at least two samples were measured. A final list of differentially expressed genes was obtained using cutoff values for both the adjusted p-value (< 0.05) and the log2 fold change (|log2FC| > 1).
Genome_build: hg19
Supplementary_files_format_and_content: raw abundance counts from htseq-counts compiled in a text file and normalized counts
|
| |
|
| Submission date |
May 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Rekin's Janky |
| E-mail(s) |
rekins.janky@med.kuleuven.be
|
| Organization name |
KU Leuven
|
| Department |
Department of Human Genetics
|
| Lab |
Laboratory of Computational Biology
|
| Street address |
Herestraat 49 box 602
|
| City |
Leuven |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL11154 |
| Series (2) |
| GSE47042 |
Discovery of the p53 targetome in MCF7 cells from RNA-seq data |
| GSE47043 |
Discovery of the p53 targetome in MCF7 cells from RNA-seq and ChIP-seq data |
|
| Relations |
| BioSample |
SAMN02146493 |
| SRA |
SRX281503 |
