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| Status |
Public on Feb 18, 2021 |
| Title |
MDA-mb231 WT rep2 |
| Sample type |
SRA |
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| Source name |
MDA-mb231 cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-mb231
genotype: wild type
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| Growth protocol |
Cells were grown in Dulbecco modified Eagle medium (DMEM - high glucose, pyruvate, GlutaMAX – Gibco® Life LifeTechnologies) supplemented with 10% foetal bovine serum (SIGMA or DUTSCHER). Cells were grown under standard conditions at 37°C in humidified incubator containing 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from about 5M cells from MDA-MB-231 WT and Ki-67 KO independent clones, that were trypsinized and centrifuged, cell pellet was treated with 1ml Trizol (Life Technologies). Then following the manufacturer’s instructions, samples were incubated 5 min at RT, added 0,2 ml Chloroform and shaked the tubes. Tubes were incubated 3 min at RT, centrifuged 12000g 15 min at 4ºC, and aqueous phase was transferred to new (RNAse free) eppendorf tubes. Followed the procedure with the RNAeasy kit from Qiagen, adding the aqueous phase to the gDNA eliminator column step from the RNAeasy kit and continued the protocol following the manufacturer’s instructions. RNA integrity was analysed on Agilent 2100 bioanalyzer.
mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads. mRNA molecules were fragmented into small pieces using fragmentation reagent after reaction a certain period in proper temperture. First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The synthesized cDNA was subjected to end-repair and then was 3’ adenylated. Adapters were ligated to the ends of these 3’ adenylated cDNA fragments. PCR was used to amplify the cDNA fragments with adapters from previous step and the products were purified with Ampure XP Beads (AGENCOURT), and dissolved in EB solution.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
DNBSEQ-G400 |
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| Data processing |
Base calling was performed with ZebraCallV2 software FastQC was used to perform quality control of the sequencing
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to GRCh38.p13 genome using STAR_2.6.0c with default parameters.
Counts of the reads per genes were calculated in the mapping step with the STAR software using the gencode vM35 gene annotations.
Differential expression analysis were performed using an Ad hoc script using the R programming software based on the Deseq2 library
Genome_build: GRCh38.p13
Supplementary_files_format_and_content: Raw counts of reads per gene
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| Submission date |
Dec 08, 2020 |
| Last update date |
Feb 18, 2021 |
| Contact name |
Daniel Fisher |
| E-mail(s) |
daniel.fisher@igmm.cnrs.fr
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| Organization name |
CNRS
|
| Department |
IGMM
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| Lab |
DFL
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| Street address |
1919 Route de Mende
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| City |
Montpellier |
| ZIP/Postal code |
34090 |
| Country |
France |
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| Platform ID |
GPL28038 |
| Series (2) |
| GSE162879 |
Next Generation Sequencing Quantitative Analysis of the transcriptomes of Wild Type and Mki67-/- MDA-mb231 (human breast adenocarcinoma) cell line |
| GSE163114 |
Ki-67 promotes carcinogenesis by enabling global transcriptional programmes |
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| Relations |
| BioSample |
SAMN17032316 |
| SRA |
SRX9652952 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4964663_wt_2_ensembl_ReadsPerGene.out.tab.gz |
390.4 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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