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| Status |
Public on May 31, 2021 |
| Title |
Cas-9 only clonal cell line, mutant-p53, experiment-3 |
| Sample type |
RNA |
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| Source name |
JH-EsoAd1 cell line derived from a patient with Barrett-associated adenocarcinoma, which has a missense c797G>A mutation in TP53 resulting in an amino acid change of G266E
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| Organism |
Homo sapiens |
| Characteristics |
cell line: JH-EsoAd1
genotype: parental (mutant-p53)
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| Treatment protocol |
The samples were collected from co-cultured cells prior to their counterparts being treated with drugs or irradiation.
SLC7A11 transfections: media was changed 24 h prior to transfection to antibiotic-free charcoal-stripped FCS. Cells were then transfected using Lipofectamine 2000 (Invitrogen, cat #11668019, Scoresby, Australia) in a pooled setting as described above. After harvesting of the cells, 2.04x 106 cells were placed in T25 flasks which resulted in 30-50% confluency after 6 h. Cells were transiently transfected with SLC7A11 siRNA (ON-TARGETplus Human SLC7A11 (23657) siRNA-SMARTpool, Dharmacon / Horizon, cat #L-007612-01-0010, Cambridge, UK) or control siRNA (ON-TARGETplus Non-targeting Pool, Dharmacon / Horizon, cat #D-001810-10-05, Cambridge, UK) to inhibit expression of SLC7A11 or not, respectively. The final siRNA concentration was 100 nM.
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| Growth protocol |
All cell lines were cultured using (phenol red free) RPMI 1640 medium supplemented with 10 % fetal bovine serum (FBS) , 50 U/ml penicillin , 50 µg/ml streptomycin , and 100 µg/ml normocin.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation from untreated cell samples at time of treatment, including DNase digestion, was performed using the miRNeasy Mini Kit (Qiagen, #217004, Chadstone, Australia) and RNase-free DNase Set (Qiagen, #79254, Chadstone, Australia) as instructed by the manufacturer.
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| Label |
FAM
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| Label protocol |
For cell line experiments, high throughput QuantStudio™ 12K Flex OpenArray® PCR custom made plates were used for miRNA profiling. These arrays were comprised of a panel of 112 miRNA probes (miRBase version 22 miRNA names, seed sequences, and miRBase accession numbers are in Supplementary file 2) that were selected based upon their abundance in OAC patient samples from our previous study on serum small extracellular vesicle associated miRNAs [94]. For each sample, 3.35 μl of RNA was reverse transcribed using a matching Custom OpenArray® miRNA RT pool (Life Technologies cat # A25630) and the TaqMan® microRNA Reverse Transcription Kit (Life Technologies cat # 4366596). cDNA Pre-amplifications were carried out with a matching Custom OpenArray® PreAmp pool (Life Technologies cat # 4485255) and TaqMan PreAmp Master Mix (Life Technologies cat # 4488593) on 7.5 μl complementary DNA (cDNA)/sample for each pool. The pre-amplified products (4 μl per sample) were diluted at the recommended 1:40 dilution with 156 μl of RNase-free ultra pure water before mixing with TaqMan OpenArray Real-Time PCR Master Mix (Life Technologies cat # 4462164) and loading onto a 384-well TaqMan OpenArray loading plate. PCR runs were performed using a QuantStudio™ 12K Flex Real-Time PCR System.
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| Hybridization protocol |
n/a
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| Scan protocol |
n/a
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| Description |
Control
Dox non-induced expt-3
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| Data processing |
Fluorescence data was exported to comma delimited text files and then analysed using R statistical software (version 3.4.3), and Microsoft Excel for Mac (version 16). The cycle threshold (Ct) value for each PCR assay was determined using the qpcR package v1.4 in R (https://cran.r-project.org/web/packages/qpcR/index.html). The relative levels of the miRNAs were determined using the formula 2^(40-Ct), and were normalized using the geometric means of the relative levels of selected House Keeping Genes (HKGs). The HKGs used in this study are included in a file on the series record named "Selected HKGs for p53-KO manuscript .xlsx".
normalized_data includes House Keeping Gene normalised miRNA relative levels
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| Submission date |
Apr 12, 2021 |
| Last update date |
May 31, 2021 |
| Contact name |
George Mayne |
| E-mail(s) |
[email protected]
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| Phone |
+61 08 8204 6088
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| Organization name |
Flinders University of South Australia
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| Department |
Surgery
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| Lab |
3d213
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| Street address |
Room 3D213, Dept of Surgery, Flinders Medical Center
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| City |
Bedford Park |
| State/province |
SA |
| ZIP/Postal code |
5042 |
| Country |
Australia |
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| Platform ID |
GPL22992 |
| Series (1) |
| GSE171965 |
Mutant p53 mediates sensitivity to cancer treatment agents in oesophageal adenocarcinoma associated with microRNA and SLC7A11 expression |
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| Supplementary data files not provided |
| Processed data are available on Series record |
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