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| Status |
Public on Apr 05, 2021 |
| Title |
cyto_MLN0128_1B |
| Sample type |
SRA |
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| Source name |
MCF7 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7 cells
rna_type: Cytosolic
treatment: MLN0128
replicate_identifier: 1B
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| Treatment protocol |
On day 0, cells were seeded in 15-cm plates. On day 2, cells were washed three times in PBS and starved in RPMI with 0.1% FBS media for 16h. This was followed by a 4h stimulation with complete media in presence of vehicle (DMSO), RMC-4627 (3nM), RMC-4745 (35nM) or MLN0128 (40nM). Cell confluence was at 80% upon harvest. Four biological replicates were generated and polysome-profiling was performed as previously described (Gandin et al. 2014).
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| Growth protocol |
MCF7 scr cells were cultured in RPMI containing 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Polysome-associated mRNA was extracted from fractions corresponding to mRNA associated with more than 3 ribosomes. RNA extraction was performed in parallel for polysome-associated and cytoplasmic RNA using Tri-reagent followed by purification using the RNeasy MinElute Cleanup Kit (Qiagen).
Sequencing libraries were constructed using the smartSeq2 protocol (Picelli et al., 2014).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
P12209_1015
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| Data processing |
The alignment (with HISAT2) and quantification (default parameters) of RNAseq reads were performed using the nf-core/rnaseq v1.2 pipeline available at https://github.com/nf-core/rnaseq (Ewels et al, 2019).
Genes with multiple loci were excluded from the analysis and only protein-coding genes were further analyzed.
Replicate 1A was excluded from the downstream analysis (it is from the same biological replicate as 1B ans was used only as a quality check).
For downstream transformation, filtering and normalization, the default methods of the anota2seq R package (Oertlin et al, 2019) were selected (Robinson & Oshlack, 2010; Ritchie et al, 2015).
Analysis of changes in mRNA abundance and changes in translational efficiencies leading to altered protein levels or buffering was carried out with anota2seq using default parameters (biological replicate was used as a batch effect in anota2seq) of the anota2seqRun function (Oertlin et al, 2019). This was implemented for the following contrasts: DMSO (full serum media) vs. starvation, MLN0128 vs. DMSO (full serum media), RMC-4627 vs. MLN0128 and RMC-4745 vs. MLN0128.
Genome_build: GRCh37
Supplementary_files_format_and_content: The processed data files are featureCounts output of the nf-core/rnaseq pipeline
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| Submission date |
Oct 03, 2019 |
| Last update date |
Mar 18, 2022 |
| Contact name |
Ola Larsson |
| E-mail(s) |
ola.larsson@ki.se
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| Phone |
+46 (0)8 517 73280
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| Organization name |
Karolinska Institutet
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| Department |
Department of oncology-pathology
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| Street address |
CCK, R8:01
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| City |
Stockholm |
| ZIP/Postal code |
171 76 |
| Country |
Sweden |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE138417 |
Selective Inhibitors of mTORC1 Activate 4EBP1 and Suppress Tumor Growth |
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| Relations |
| BioSample |
SAMN12911272 |
| SRA |
SRX6949868 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4107735_P12209_1015_S15_bothLanes_R1_001.sorted_gene.featureCounts.txt.gz |
5.0 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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