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Sample GSM4081797 Query DataSets for GSM4081797
Status Public on Aug 19, 2021
Title Replicate 1 charging not5delta 3418
Sample type RNA
 
Channel 1
Source name cultivated not5delta charged cells
Organism Saccharomyces cerevisiae
Characteristics genotype/variation: not5D (isogenic except not5::KANMX4)
Growth protocol Cells were cultivated as described in: Pansenko et al. Co-translational assembly of proteasome subunits in NOT1-containing assemblysomes, Nature Structural & Molecular Biologyvolume 26, pages110–120 (2019).
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 tRNASer3, human tRNAHis1) were added to 5 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 1h at RT in the presence of 15% DMSO (Sigma-Aldrich).
 
Channel 2
Source name cultivated not5delta uncharged cells
Organism Saccharomyces cerevisiae
Characteristics genotype/variation: not5D (isogenic except not5::KANMX4)
Growth protocol Cells were cultivated as described in: Pansenko et al. Co-translational assembly of proteasome subunits in NOT1-containing assemblysomes, Nature Structural & Molecular Biologyvolume 26, pages110–120 (2019).
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 tRNASer3, human tRNAHis1) were added to 5 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 1h at RT 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 2.
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, human tRNASer3, E. coli tRNATyr2, human tRNAHis1) for individual blocks.
4. Normalized Cy3 532/Atto635 ratios for individual blocks were averaged, representing final Cy3 532/Atto635 ratios.
 
Submission date Sep 17, 2019
Last update date Aug 19, 2021
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 GPL27470
Series (1)
GSE137567 Not4 and Not5 modulate translation elongation by Rps7A ubiquitination, Rli1 moonlighting, and condensates that exclude eIF5A

Data table header descriptions
ID_REF
VALUE Normalized ratio (Cy3 532/Atto635)

Data table
ID_REF VALUE
E.c. Tyr-2 1.002037005604688
His1h 0.9933399858344368
E.c. Lys-2 0.9731379392639179
Ser3h 0.0
Arg1y 1.136658448674142
Arg2y 0.9345057968323985
Arg3y 1.1848721870757388
Arg4y 1.1345648711091654
His1y 0.0
Lys1y 0.6979962950163585
Lys2y 0.4229368869011325
Asp1y 0.8754326094896805
Asp2y 0.8782949646741476
Glu1y 0.6079949647741115
Asn1y 0.9963258264783186
Cys1y 1.1082857136793909
Gln1y 0.8624278555670174
Ser1y 0.9985406502000602
Ser2y 1.1849623970579037
Ser3y 0.56760505296733

Total number of rows: 68

Table truncated, full table size 1 Kbytes.




Supplementary file Size Download File type/resource
GSM4081797_charging_not5delta_rep1.txt.gz 97.4 Kb (ftp)(http) TXT
Processed data included within Sample table

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