|
| Status |
Public on Mar 22, 2022 |
| Title |
Melan-a_siScr_24hrs_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Melan-a cells, siScr, 24h
|
| Organism |
Mus musculus |
| Characteristics |
cell line: Melan-a cells
strain: C57BL/6
sirna: siScr
Sex: female
rin: 9.8
passage: 54
|
| Treatment protocol |
Melan-a cells were transfected with siDicer or siScr using lipofectamin (Invitrogen 11668019) and magnetofectamin (OZ bioscience CM20200) following manufacturer instructions. For each well, 2 ul of lipofectamin, 1 ul of magnetofectamin and 50 pmol of siRNA diluted in 100 ul Optimem (Gibco) and 900 ul F12 medium (Gibco 21765) without antibiotics were added to the cells. Plates were incubated at 37°C on a magnetic plate which was remove 20 minutes later. Six hours later, medium was replaced by complete F12 medium. Cell lysates were collected 24 hours later by scratching in Qiazol reagent as indicated in miRNeasy Kit (Qiagen, #217004) protocol.
|
| Growth protocol |
The mouse melanocyte Melan-a cell line was provided by D. Bennett (Bennett et al, 1987) and cultured in F12 medium (Gibco 21765) supplemented with 10% fetal bovine serum (Eurobio CVFSVF00-01), 1% penicillin/streptomycin (Gibco 15140-122), and 200 nM TPA (Sigma P8139). For transfection experiements, 150 000 Melan-a cells were seeded in a well of 12-well plate 24 hours before transfection. Cells were grown at 37°C in a humidified atmosphere containing 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from Melan-a cells was extracted using the miRNeasy Kit (Qiagen, #217004).
|
| Label |
biotin
|
| Label protocol |
Sample preparation and hybridization were performed by the Genomic platform of the Institut Curie. Mouse ClariomD arrays were hybridized according the recommendations of Affymetrix (Santa Clara, CA, United States) using the WT PLUS protocol and labelling and hybridization kits from Affymetrix. Total RNA (100 ng) was processed in parallel with an external Universal Mouse Reference RNA to verify the robustness of the data. The mean yield of labelled DNA was 9.75 µg (min: 8.15 µg; max: 11.65 µg).
|
| |
|
| Hybridization protocol |
Affymetrix GeneChip® Mouse ClariomD microarrays were hybridized with 4.7 µg labelled DNA.
|
| Scan protocol |
The microarrays were scanned using a GeneChip Scanner 3000 7G System (Affymetrix, Santa Clara, CA, United States)
|
| Data processing |
Microarray analysis was performed by Genosplice Technology. Quality controls were performed using Expression Console metrics (Affymetrix). Further analysis and visualization were performed with EASANA (GenoSplice Technology, www.genosplice.com), using FAST DB 2016_1 annotations as already described {Tury, 2018 #3315;Sharma, 2017 #3316;Schaller, 2017 #3317}. Data were normalized by quantile normalization. Background corrections were made with antigenomic probes selected as previously described. Only probes targeting exons annotated from FAST DB transcripts were selected to ensure that we focused on well-annotated genes for which mRNA sequences were present in public databases. Low-quality probes (e.g., probes labeled by Affymetrix as “cross-hybridizing”) and those with a low signal intensity (relative to antigenomic background probes with the same GC content) were removed from the analysis. Only probes with a detection above background (DABG), p ≤ 0.05, in at least half the arrays were considered for statistical analysis. Only genes expressed in at least one of the compared conditions compared were analyzed. Expression was defined as a DABG p ≤ 0.05 for at least half of the gene probes. A paired Student’s t-test was used to compare gene-expression intensities between the various biological replicates.
|
| |
|
| Submission date |
Mar 24, 2019 |
| Last update date |
Mar 23, 2022 |
| Contact name |
Juliette BERTRAND |
| E-mail(s) |
[email protected]
|
| Organization name |
Institut Curie
|
| Department |
CNRS UMR3347, INSERM U1021
|
| Street address |
Bat 110, Centre universitaire
|
| City |
ORSAY Cedex |
| ZIP/Postal code |
91405 |
| Country |
France |
| |
|
| Platform ID |
GPL20258 |
| Series (2) |
| GSE128755 |
Gene expression data from Melan-a cells expressing reduced level of Dicer and control Melan-a cells [24 hours] |
| GSE128757 |
Melan-a cells expressing reduced level of Dicer and control Melan-a cells |
|
