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Sample GSM4081802 Query DataSets for GSM4081802
Status Public on Aug 19, 2021
Title Replicate 2 abundance not5delta 3418
Sample type RNA
 
Channel 1
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 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 wild-type cells - uncharged tRNA
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 2 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 0.9804345070776271
His1h 1.0205136949854474
E.c. Lys-2 0.9531108116367017
Ser3h 0.9820751497888662
Arg1y 1.5473635129033778
Arg2y 0.9513606737910408
Arg3y 1.2987056692866754
Arg4y 0.6099589394829983
His1y 0.9262172575696819
Lys1y 0.594383815846502
Lys2y 1.3783441062257944
Asp1y 1.6438294581667627
Asp2y 1.7618562904312305
Glu1y 1.5863351599276916
Asn1y 1.2720981767179758
Cys1y 0.37001564525769703
Gln1y 0.3129771299965972
Ser1y 0.7468219209627972
Ser2y 1.536621509758334
Ser3y 2.147413679897942

Total number of rows: 68

Table truncated, full table size 1 Kbytes.




Supplementary file Size Download File type/resource
GSM4081802_abundance_not5delta_rep2.txt.gz 116.3 Kb (ftp)(http) TXT
Processed data included within Sample table

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