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Sample GSM6706309 Query DataSets for GSM6706309
Status Public on Apr 12, 2023
Title RPS26dC, Glucose starvation, Light fraction, replicate 2
Sample type SRA
Source name kidney
Organism Homo sapiens
Characteristics tissue: kidney
cell line: HEK293 TAF1 ts mutant cells
genotype: RPS26dC
treatment: Glucose starvation
Treatment protocol Untreated and 4 hours glucose starved [glucose-free media (11966025, Gibco) with 10% dialyzed FBS] WT and RPS26dC cells were incubated with 100 µg/ml Cycloheximide (CHX, Sigma) for 5 min
Growth protocol cells were grown at 37oC in a humidified incubator with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO, Life Technologies, Thermo Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (GIBCO), 100 units/mL penicillin and 100 ug/mL streptomycin.
Extracted molecule total RNA
Extraction protocol Cells were washed twice with cold buffer containing 20 mM Tris pH 8, 140 mM KCl, 5 mM MgCl2 and 100 µg/ml CHX. The cells were collected and lyzed with 500 µl of the same buffer containing 0.5% Triton, 0.5% DOC, 1.5 mM DTT, 150 units RNAse inhibitor (Eurx) and 5 µl of protease inhibitor (Sigma). The lyzed samples were centrifuged at 12,000g at 4˚C for 5 min. The cleared lysates were loaded onto a 10–50% sucrose gradient and centrifuged at 38,000 RPM in an SW41 rotor for 105 min at 4˚C. Gradients were fractionated and the optical density at 254 nm was continuously recorded using the ISCO absorbance detector UA-6. The collected samples were merged to create the fractions: Light polysome (2-5 ribosomes) and Heavy (6+ ribosomes). The RNA of these fractions and the Input samples were isolated using Trizol and Direc-Zol RNA mini-prep kits (Zymo Research).
Equivalent RNA concentrations were taken from each fraction for the library preparation for MARS-Seq
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2500
Description RPS26dC, Glucose starvation, Light fraction, replicate 2
Data processing Polysome profile reads were aligned to the human reference genome (hg38) using Bowtie2
The number of reads per gene was counted from the uniquely aligned reads in genecode (GRCh38.103) genomic regions using featureCounts
Normalization between samples was performed using the DESeq2 package in the R statistical programming language
Replicates similarities were tested using principal component analysis (PCA) and Pearson correlations and plotted using ggplot2 in R
The normalized gene read counts table (DESeq2 output table) was filtered to include only genes in high expression: only genes that the sample with the least number of reads per gene has > 32 reads were used for downstream analysis.
The normalized counts were used to calculate translation efficiency (TE)
The normalized counts were used to calculate translation efficiency (TE) (See manuscript: Havkin-Solomon et al.)
Assembly: hg38
Supplementary files format and content: Content: Gene reads coverage. Based on human gencode genes. Result of featureCount (txt, tabular format). Non-normalized.
Supplementary files format and content: File name: RPS26_project_Feb2021.gene_expression_levels.protein_coding_genes.with_gene_symbol_and_biotype.diff_lanes_sum_togther.RPS26_project.txt
Submission date Nov 03, 2022
Last update date Apr 12, 2023
Contact name Tal Havkin-Solomon
Phone +972-508311789
Organization name Weizmann Institute of Science
Department Biomolecular Sciences
Lab Rivka Dikstein
Street address 234 Herzl
City Rehovot
ZIP/Postal code 7610001
Country Israel
Platform ID GPL16791
Series (1)
GSE217180 Translation regulation of specific mRNAs by RPS26 C-terminal RNA-binding tail integrates energy metabolism and AMPK-mTOR signaling
BioSample SAMN31582344
SRA SRX18154946

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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