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Sample GSM4081795 Query DataSets for GSM4081795
Status Public on Aug 19, 2021
Title Replicate 1 charging not4delta 3417
Sample type RNA
 
Channel 1
Source name cultivated not4delta charged cells
Organism Saccharomyces cerevisiae
Characteristics genotype/variation: not4D (isogenic except not4::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 not4delta uncharged cells
Organism Saccharomyces cerevisiae
Characteristics genotype/variation: not4D (isogenic except not4::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 0.0
His1h 1.006599484816958
E.c. Lys-2 0.0
Ser3h 1.0679164673920274
Arg1y 0.6636519005641979
Arg2y 0.7165515443432006
Arg3y 0.650076516075268
Arg4y 0.6483537132168228
His1y 0.5083141376461243
Lys1y 0.8827402821532266
Lys2y 0.424686788681937
Asp1y 0.5481606760458159
Asp2y 0.5788218364779827
Glu1y 0.469787072451852
Asn1y 0.5445405848607048
Cys1y 0.6230403484104717
Gln1y 0.6327510088437297
Ser1y 0.42681333930839777
Ser2y 0.5858456705327705
Ser3y 0.5703577484781199

Total number of rows: 68

Table truncated, full table size 1 Kbytes.




Supplementary file Size Download File type/resource
GSM4081795_charging_not4delta_rep1.txt.gz 119.6 Kb (ftp)(http) TXT
Processed data included within Sample table

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