|
| Status |
Public on Jan 01, 2017 |
| Title |
U251-BMI1-shRNA |
| Sample type |
RNA |
| |
|
| Source name |
whole cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U251
phenotype: reduced BMI1 expression
|
| Treatment protocol |
cells were stably transduced with a lentiviral vector containing the shRNA sequence of BMI1, USP22 and control
|
| Growth protocol |
U251MG was grown in the DMEM supplemented with 10% FBS (GIBCO)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by mirVanaTM RNA Isolation Kit (Applied Biosystem p/n AM1556 ) following the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 μg RNA using the One-Color Low RNA Input Linear Amplification PLUS 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.
|
| |
|
| Hybridization protocol |
0.6 μg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 22.5μl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers' instructions. On completion of the fragmentation reaction, 22.5μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human Gene Expression 8x60K v2 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 immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 4x180k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression after reducing BMI1 expression
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.1.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities as the raw data. Raw data were normalized in quantile algorithm with Genespring 13.0(Agilent). Probe that at least 1 out of 2 samples flagged as Detected were maintained.
|
| |
|
| Submission date |
Oct 27, 2016 |
| Last update date |
Apr 23, 2018 |
| Contact name |
Jiangbing zhou |
| Organization name |
Yale university
|
| Street address |
310 Cedar Street, Ste LH 412A
|
| City |
Newhaven |
| ZIP/Postal code |
06510 |
| Country |
USA |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE89239 |
The deubiquitinase USP22 stabilizes BMI1 polycomb ring finger oncoprotein to confer cancer stem cell traits and glioblastoma progression |
|
| Relations |
| Reanalyzed by |
GSE113533 |
