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Sample GSM5727352 Query DataSets for GSM5727352
Status Public on Jan 31, 2023
Title abundance not1 degron vs control strain replicate 1
Sample type RNA
 
Channel 1
Source name cultivated cells
Organism Saccharomyces cerevisiae
Characteristics cell line: MY13523 MATa leu2-3,112 HIS3,15 ura3-1::pADH1-OsTIR1-URA3 ADE2 trp1-1 can1-100 not1::NOT1-AID-Myc6-NATMX4
Treatment protocol Cells were treated with auxin (3-indoleacetic acid, Sigma-Aldrich I2886, stock solution at 250 mM in EtOH) at 1mM final for 15 min to exponentially growing cells diluted to OD600 0.3
Growth protocol Cells were cultivated in glucose rich medium (YPD),grown over night until OD600 0.8, then treated
Cell were grown independently for each biological experiment
Extracted molecule total RNA
Extraction protocol Total RNA for total tRNA arrays was extracted using TriReagent (Sigma-Aldrich) and for arrays to determine the charged fraction with acidic phenol (pH 4.5) according to manufacturer's protocol
Label Cy3
Label protocol 1. Equal amounts of in vitro synthesized tRNA standards (4 pmol each; E. coli tRNALys2, E. coli tRNATyr2, human tRNAHis1, human tRNASer3) were added to 2 µg extracted total RNA prior to deacetylation.
2. For abundance arrays total RNA was deacylated for 45 min at 37°C with 100 mM Tris-HCl (pH 9); For tRNA charging arrays total RNA was oxidized with periodate and thereafter treated for 45 min at 37°C with 100 mM Tris-HCl (pH 9) for tRNA deacetylation; tRNA standards were added prior the deacylation step.
3. Cy3 or Atto653 containing oligonucleotides (Microsynth) were ligated to tRNA 3'-CCA ends using 20 U/μl T4 DNA ligase (New England Biolabs) for 16h at 16°C in the presence of 15% DMSO (Sigma-Aldrich).
 
Channel 2
Source name cultivated cells
Organism Saccharomyces cerevisiae
Characteristics cell line: MY13472 MATa leu2-3,112 HIS3,15 ura3-1::pADH1-OsTIR1-URA3 ADE2 trp1-1 can1-100
Treatment protocol Cells were treated with auxin (3-indoleacetic acid, Sigma-Aldrich I2886, stock solution at 250 mM in EtOH) at 1mM final for 15 min to exponentially growing cells diluted to OD600 0.3
Growth protocol Cells were cultivated in glucose rich medium (YPD),grown over night until OD600 0.8, then treated
Cell were grown independently for each biological experiment
Extracted molecule total RNA
Extraction protocol Total RNA for total tRNA arrays was extracted using TriReagent (Sigma-Aldrich) and for arrays to determine the charged fraction with acidic phenol (pH 4.5) according to manufacturer's protocol
Label Atto635
Label protocol 1. Equal amounts of in vitro synthesized tRNA standards (4 pmol each; E. coli tRNALys2, E. coli tRNATyr2, human tRNAHis1, human tRNASer3) were added to 2 µg extracted total RNA prior to deacetylation.
2. For abundance arrays total RNA was deacylated for 45 min at 37°C with 100 mM Tris-HCl (pH 9); For tRNA charging arrays total RNA was oxidized with periodate and thereafter treated for 45 min at 37°C with 100 mM Tris-HCl (pH 9) for tRNA deacetylation; tRNA standards were added prior the deacylation step.
3. Cy3 or Atto653 containing oligonucleotides (Microsynth) were ligated to tRNA 3'-CCA ends using 20 U/μl T4 DNA ligase (New England Biolabs) for 16h at 16°C in the presence of 15% DMSO (Sigma-Aldrich).
 
 
Hybridization protocol 1. Fluorescently labeled tRNAs were hybridized on the microarrays for 16 h at 60°C in a Hyb4 microarray hybridization system (Digilab) supplemented with polyA (0.17 mg/ml) and salmon sperm DNA (0.17 mg/ml).
2. Microarrays were washed once in 2× SSC/0.1% SDS (50°C), once in 1× SSC/0.1% SDS (42°C) and then three times in 0.1× SSC (42°C).
3. Arrays were dried and stored in the dark at 4°C.
Scan protocol Arrays were scanned with a GenPIX 4200A (Molecular Devices) instrument.
Description Biological replicate 1 of 3.
Data processing 1. TIF-files of scanned arrays were analyzed using the GenePix Pro 7 (Molecular Devices) software.
2. Ratios of Cy3/Atto635 fluorescence signals were calculated for individual spots based on mean fluorescence signal (background fluorescence substraction).
3. Cy3 532/Atto635 ratios were normalized to Cy3 532/Atto635 values of tRNA standards (e.g. median Cy3 532/Atto635 ratios of the four tRNA standards E. coli tRNALys2, yeast tRNAAla5, E. coli tRNATyr2, Yeast tRNAPhe3) for individual blocks.
4. Normalized Cy3 532/Atto635 ratios for individual blocks were averaged, representing final Cy3 532/Atto635 ratios.
 
Submission date Dec 10, 2021
Last update date Feb 02, 2023
Contact name Zoya Ignatova
E-mail(s) zoya.ignatova@chemie.uni-hamburg.de
Organization name Universitaet Hamburg
Street address Martin -Luther-King-Platz 6
City Hamburg
ZIP/Postal code 20146
Country Germany
 
Platform ID GPL28469
Series (1)
GSE190658 Not1 and Not4 inversely determine mRNA solubility that sets the dynamics of co-translational events

Supplementary file Size Download File type/resource
GSM5727352_Abundance_rep1.tsv.gz 121.8 Kb (ftp)(http) TSV
Processed data are available on Series record

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