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Sample GSM4107731 Query DataSets for GSM4107731
Status Public on Apr 05, 2021
Title cyto_RMC4627_1B
Sample type SRA
Source name MCF7 cells
Organism Homo sapiens
Characteristics cell line: MCF7 cells
rna_type: Cytosolic
treatment: RMC4627
replicate_identifier: 1B
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).
Growth protocol MCF7 scr cells were cultured in RPMI containing 10% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2.
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).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Description P12209_1011
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 (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
Submission date Oct 03, 2019
Last update date Mar 18, 2022
Contact name Ola Larsson
Phone +46 (0)8 517 73280
Organization name Karolinska Institutet
Department Department of oncology-pathology
Street address CCK, R8:01
City Stockholm
ZIP/Postal code 171 76
Country Sweden
Platform ID GPL24676
Series (1)
GSE138417 Selective Inhibitors of mTORC1 Activate 4EBP1 and Suppress Tumor Growth
BioSample SAMN12911276
SRA SRX6949864

Supplementary file Size Download File type/resource
GSM4107731_P12209_1011_S11_bothLanes_R1_001.sorted_gene.featureCounts.txt.gz 5.0 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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