|
| Status |
Public on Jun 15, 2020 |
| Title |
si-eIF3e/si-ctrl replicate 1-2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
cultivated cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-10A
treatment: si-control
|
| Treatment protocol |
The cells were treated with 20nM si-control or si-eIF3e for 72h.
|
| Growth protocol |
MCF-10a cells were cultured in DMEM/F12 medium with 0.5mg/mL hydrocortisone, 10µg/mL insulin, 20ng/mL EGF, 5% serum, 1X Pen/Strep, and incubate in 37℃ with 5% CO2.
Cell were grown independently for each biological experiment and checked frequently for mycoplasma contamination.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA for total tRNA arrays was extracted using TriReagent (Sigma-Aldrich) 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, yeast tRNAAla5, yeast tRNAPhe3) were added to 2 µg extracted total RNA prior to deacetylation.
2. For charging arrays total RNA was deacylated for 45 min at 37°C with 100 mM Tris-HCl (pH 9); For tRNA abundance 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 teh deacyaltion 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 cells
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: siRNA for eIF3e
cell line: MCF-10A
|
| Treatment protocol |
The cells were treated with 20nM si-control or si-eIF3e for 72h.
|
| Growth protocol |
MCF-10a cells were cultured in DMEM/F12 medium with 0.5mg/mL hydrocortisone, 10µg/mL insulin, 20ng/mL EGF, 5% serum, 1X Pen/Strep, and incubate in 37℃ with 5% CO2.
Cell were grown independently for each biological experiment and checked frequently for mycoplasma contamination.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA for total tRNA arrays was extracted using TriReagent (Sigma-Aldrich) 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, yeast tRNAAla5, yeast tRNAPhe3) were added to 2 µg extracted total RNA prior to deacetylation.
2. For charging arrays total RNA was deacylated for 45 min at 37°C with 100 mM Tris-HCl (pH 9); For tRNA abundance 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 teh deacyaltion 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 1 Cy3 vs 2 of 3 Atto
|
| 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 median fluorescence signal (background fluorescence subtraction).
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; detailed protocol for normalization using the dye control slide is published in: https://www.protocols.io/view/microarray-based-quantification-of-cellular-trnas-hfcb3iw or in : Dittmar KA, Mobley EM, Radek AJ, Pan T. (2004) Exploring the regulation of tRNA distribution on the genomic scale. J Mol Biol 12, 31-47
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| |
|
| Submission date |
May 01, 2020 |
| Last update date |
Jun 16, 2020 |
| 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) |
| GSE149697 |
eIF3 associates with 80S ribosomes to promote translation elongation, mitochondrial homeostasis, and muscle health |
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