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| Status |
Public on Feb 22, 2019 |
| Title |
RNA from ACC-Meso-4 expressing shRNA targeting CBX6_2 |
| Sample type |
RNA |
| |
|
| Source name |
ACC-Meso-4, CBX6 shRNA, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
shRNA: shCBX6
cell line: Human malignant pleural mesothelioma cell line. Japanese, Male, 59 years
|
| Treatment protocol |
Lentiviruses were produced in 293T cells transfected with packaging plasmids (Sigma-Aldrich) and shRNA constructs by using lipofectamine LTX (Invitrogen). Each shRNA was introduced by lentivuruses into ACC-Meso-4 cells and stable knockdowns in ACC-Meso-4 were selected with 1 µg/ml puromycin in culture medium for 3 weeks.
|
| Growth protocol |
ACC-Meso-4 cells were cultured with RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum. The cultures were incubated at 37 ºC in a humidified incubator with 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared using TRIZOL reagent (Invitrogen). The quality of RNA samples were assessed by RNA 6000 nano kit (Agilent Bioanalyzer 2100).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low RNA Input Fluorescent Linear Amplification kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >6.0 pmol Cy3/ug 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 manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent-039494 Whole Mouse Genome Microarray 8x60K 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), then dried by raise it slowly from buffer 2.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2565BA) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green. Scan mode is eXtended Dynamic range Scan mode and PMT is set to 10% and 100%).
|
| Description |
Gene expression in ACC-Meso-4 expressing shRNA targeting CBX6_2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 026655_D_20120201). Further analysis were performed by GeneSpring GX 12.1 software (Agilent Technologies). Normalization was performed as follows: (i) intensity-dependent Lowess normalization; (ii) data transformation, with measurements set to ≤0.01; (iii) per-chip 75th-percentile normalization of each array; and, (iv) per-gene: normalized to the median of each gene.
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| |
|
| Submission date |
Feb 15, 2019 |
| Last update date |
Feb 22, 2019 |
| Contact name |
Takumi Nishiuchi |
| E-mail(s) |
[email protected]
|
| Organization name |
Kanazawa University
|
| Street address |
13-1 Takaramachi
|
| City |
Kanazawa |
| ZIP/Postal code |
920-0934 |
| Country |
Japan |
| |
|
| Platform ID |
GPL21061 |
| Series (1) |
| GSE126605 |
Gene expression of mesothelioma cell line ACC-Meso-4 treated with shRNA targeting chromobox (CBX) 6, 7, and 8. |
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