|
| Status |
Public on Apr 01, 2022 |
| Title |
Ies6_siRNA_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Ies6 siRNA in RD cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: RD
cell type: human rhabdomyosarcoma cells
genotype/variation: Ies6 knockdown
|
| Treatment protocol |
RD cells were transfected with siRNAs using Lipofectamine RNAiMAX for 2 days
|
| Growth protocol |
RD cells were cultured with a Dulbecco Modified Eagle medium supplemented with 20% foetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were extracted using RNAiso plus, and purified using NucleoSpin RNA Plus.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1ug Total RNA using the Low Input Quick Amp Labeling Kit(Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
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| |
|
| Hybridization protocol |
0.6ug of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 mouse GE 8x60K Microarray Ver2.0 (Agilent) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G2600D) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 11.5.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
Normalized signal values were calculated by the following method using the Feature Extraction Software: (1) control probes were excluted by setting control type to 0 (2) averaged sigal intensity values were calculated after excluding the top and bottom 2% of Processed Signal(3) scaling factors (SF) were calculated using the formula SF=target signal constant (2500) / the trimmed mean values. (4) normalized singal intensities Scale Signal were obtained by multiplying the SF value by the individula probe signal values
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| |
|
| Submission date |
Mar 25, 2021 |
| Last update date |
Apr 01, 2022 |
| Contact name |
Tsuyoshi Morita |
| E-mail(s) |
tsuyo@wakayama-med.ac.jp
|
| Organization name |
Wakayama Medical University
|
| Department |
Department of Biology
|
| Street address |
Mikazura 580
|
| City |
Wakayama |
| ZIP/Postal code |
6410011 |
| Country |
Japan |
| |
|
| Platform ID |
GPL20844 |
| Series (1) |
| GSE169681 |
Knockdown of Ino80 complex subunits Actr5, Ies6, and Ino80 in RD cells |
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