|
| Status |
Public on Aug 19, 2021 |
| Title |
Replicate 2 charging WT 3415 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cultivated wild-type cells - charged tRNA
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype/variation: MY3415 [BY4739 (Euroscarf) MATalpha his3 leu2 ura3 lys2]
|
| 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: MY3415 [BY4739 (Euroscarf) MATalpha his3 leu2 ura3 lys2]
|
| 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 |
|
