|
| Status |
Public on May 19, 2015 |
| Title |
shRNA-depleted HeLa Cells-non-polysomal-REP1 |
| Sample type |
RNA |
| |
|
| Source name |
shRNA-depleted HeLa Cells-non-polysomal
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Adherent HeLa cells
genotype: TIA-depleted
rna fraction: Non-polysomal
|
| Growth protocol |
Adherent HeLa cells were cultured under standard conditions. The TIA1 and TIAR shRNA-pSUPER constructs were generated with the pSUPER RNA interference (RNAi) System (OligoEngine, USA). Briefly, to knock-down TIA1 and TIAR, we have followed previously described methods (OligoEngine, USA) to generate TIA1 and TIAR short hairpin RNA (shRNA) pSUPER or empty pSUPER constructs from pSUPER.retro vectors. Sequences coding for short hairpin RNAs were inserted as double-stranded oligos followed by a short spacer, the reverse complement of the sense strand and five thymidines as an RNA polymerase III transcriptional stop signal into pSUPER vectors using the BglII and XhoI sites. The target sequences for the TIA1 and TIAR genes were 5'-AAGCTCTAATTCTGCAACTCT-3' and 5'-AACCATGGAATC AACAAGGAT-3', respectively. The configuration of the constructs was verified by DNA sequencing. The TIA1 and TIAR shRNA pSUPER constructs and the empty pSUPER vector as control were transfected into the cell line HeLa (1 μg of DNA/1×106 cells). Stable cell lines expressing empty or TIA1 and/or TIAR shRNA-pSUPER constructs were selected by plating for 3 weeks according to the manufacturer’s instructions (OligoEngine, USA). Clonal lines were isolated with cloning cylinders and verified by Western blotting.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from above HeLa cell lines was isolated and purified by using RNeasy kit (Qiagen). The quality of the RNAs was tested using the Agilent 2100 Bioanalyzer RNA Nano Chip
|
| Label |
Cy3
|
| Label protocol |
Total RNA (200 ng) was amplified using One Color Low Input Quick Amp Labeling Kit (Agilent Technologies) and purified with RNeasy Mini Kit (Qiagen). Preparation of probes and hybridization was performed as described One-Color Microarray Based Gene Expression Analysis Manual Ver. 6.5, Agilent Technologies.
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| |
|
| Hybridization protocol |
The samples were placed on ice and quickly loaded onto arrays, hybridized at 65ºC for 17 hours in a Hybridization oven rotator and then washed in GE wash buffer 1 at room temperature (1 minute) and in GE Wash Buffer 2 at 37ºC (1 minute).
|
| Scan protocol |
Images were captured with an Agilent Microarray Scanner and spots quantified using Feature Extraction Software (Agilent Technologies).
|
| Description |
shRNA-depleted HeLa Cells-non-polysomal-REP1
|
| Data processing |
Raw intensities were background-corrected by normexp method with an offset of 50. Background-corrected signals were log2 transformed and normalized by quantile adjustment as described in limma package of bioconductor.
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| |
|
| Submission date |
Jan 30, 2014 |
| Last update date |
May 21, 2015 |
| Contact name |
Juan Carlos Oliveros |
| Organization name |
CNB, CSIC
|
| Street address |
Darwin 3
|
| City |
Cantoblanco |
| State/province |
Madrid |
| ZIP/Postal code |
28049 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE54540 |
TIA proteins drives global and specific translational repression of genes linked to the cell-cycle progression and DNA replication/repair |
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