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| Status |
Public on Apr 20, 2024 |
| Title |
AntagomiR-141 treated cells |
| Sample type |
SRA |
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| Source name |
Corneal epithelium
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| Organism |
Oryctolagus cuniculus |
| Characteristics |
tissue: Corneal epithelium
cell line: RCE1(5T5)
cell type: Corneal epithelium
treatment: antagomiR-141 transfection
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| Treatment protocol |
For miRNA inhibition experiments, RCE1(5T5) cells were seeded at previously indicated densities in the presence of 3T3 feeder cells. On day 4 of cell culture, feeder cells were detached from the culture plate using 0.02 % (w/v) EDTA in PBS wash. After that, epithelial cells were transfected with miRVana (Life Technologies, Carlsbad, Ca., USA) antagomiR-141-3p (MH10860) for miR-141-3p inhibition at 30 nM concentration. Transfection was made with Lipofectamine RNAiMAX (Invitrogen, Carlsbad, Ca., USA). The lipid-RNA complexes were added to the culture media according to the manufacturer's instructions. 24 hours after, fresh culture media was added, and 48 hours after transfection, cultures were washed with PBS, and RNA was subsequently isolated.
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| Growth protocol |
Cells were seeded at 2.7 x 103 cells/cm 2 together with mitomycin C-treated 3T3 cells plated at 2.2 x 104 cells/cm2, as previously described (Castro-Muñozledo, 1994, J Cell Sci 107, 2343-2351). Cells were cultured using the mixture DMEM-F12-Ham (3:1) supplemented with 5% fetal calf serum, 5 µg/ml insulin, 5 µg/ml transferrin, 0.4 µg/ml hydrocortisone, 2×10−9 M triiodothyronine, 1×10−10 M cholera toxin, 24.3 mg/l adenine and 10 ng/ml epidermal growth factor (EGF). Medium was changed every other day. Cultures were maintained at 36° C in a 10% CO2 and 90% air humidified atmosphere.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Before RNA isolation, the 3T3-feeder cells were removed by a 5-min incubation with 0.02% (w/v) EDTA in PBS. Samples were washed twice with PBS; afterwards, total RNA was isolated at 4th, 6th and 10th days in culture, and 48 hrs after transfection of antagomiR-141, with Trizol reagent according to the instructions of the manufacturer, then resuspended in DNase/RNase free water.
cDNA libraries were generated using the Illumina TruSeq RNA Sample Preparation Kit according to the manufacturer’s instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Ant-141
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| Data processing |
Quality evaluation of raw reads, adapter removal and pairing of reads were carried out using the Geneious workflow software (Geneious version 9.1.8).
Paired reads were mapped to the reference genome OryCun2.0; GCF_000003625.3, using the Geneious for RNAseq mapper.
Differential expression (DE) analysis was carried out using the Geneious tools, calculating expression measures TPM, RPKM and FPKM normalized by the Median of Gene Expression Ratios method through the forth samples.
Assembly: oc2 GCF_000003625.3
Supplementary files format and content: tab-delimited text file including expression values for each sample
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| Submission date |
Apr 15, 2024 |
| Last update date |
Apr 20, 2024 |
| Contact name |
Federico Castro-Muñozledo |
| E-mail(s) |
fcastro@cell.cinvestav.mx
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| Organization name |
CINVESTAV
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| Department |
Cell Biology
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| Lab |
28
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| Street address |
Av Instituto Politecnico Nacional 2508, San Pedro Zacatenco
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| City |
Mexico City |
| ZIP/Postal code |
07360 |
| Country |
Mexico |
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| Platform ID |
GPL16405 |
| Series (1) |
| GSE264014 |
miR-141-3p regulates the expression of corneal epithelial differentiation |
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| Relations |
| BioSample |
SAMN40972327 |
| SRA |
SRX24267354 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8208825_AntagomiR-141_transfected_RCE1_cells_transcriptome.txt.gz |
502.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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