|
| Status |
Public on Aug 28, 2022 |
| Title |
shLuc vs. shBLCAP cells |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
shLuc cells, harvested after several passages.
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK-293 (CRL-1573)
genotype: shLuc cells
|
| Treatment protocol |
shRNA against firefly Luciferase or human BLCAP gene were transducted into HEK293 cells by lentivirus infection.
|
| Growth protocol |
HEK293 cells were cultured in minimum essential media (MEM) supplemented with 10% FBS, 100 U/ml of penicillin, 0.1 mg/ml streptomycin and 1 mM sodium pyruvate at 37°C in a humidified 5% CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
RNA from Luc was labeled by Cy3 and RNA from BC10-5 was labeled by Cy5. 0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and labeled with Cy3 or Cy5 (CyDye, Agilent Technologies, USA) during the in vitro transcription process.
|
| |
|
| Channel 2 |
| Source name |
BLCAP knockdown cells, harvested after several passages.
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK-293 (CRL-1573)
genotype: BLCAP knockdown
|
| Treatment protocol |
shRNA against firefly Luciferase or human BLCAP gene were transducted into HEK293 cells by lentivirus infection.
|
| Growth protocol |
HEK293 cells were cultured in minimum essential media (MEM) supplemented with 10% FBS, 100 U/ml of penicillin, 0.1 mg/ml streptomycin and 1 mM sodium pyruvate at 37°C in a humidified 5% CO2 incubator.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
RNA from Luc was labeled by Cy3 and RNA from BC10-5 was labeled by Cy5. 0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and labeled with Cy3 or Cy5 (CyDye, Agilent Technologies, USA) during the in vitro transcription process.
|
| |
|
| |
| Hybridization protocol |
0.3 μg of Cy-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60°C for 30 minutes. Correspondingly fragmented labeled cRNA is then pooled and hybridized to a Agilent SurePrint G3 Human GE 8×60K Microarray (Agilent Technologies, USA) at 65°C for 17 h. After hybridization, slides were washed and dried by nitrogen gun blowing.
|
| Scan protocol |
Microarrays are scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3 and 625 nm for Cy5.
|
| Data processing |
Scanned images are analyzed by Feature extraction 10.5.1.1 software (Agilent Technologies, USA), an image analysis and normalization software is used to quantify signal and background intensity for each feature, substantially normalized the data by rank-consistency-filtering LOWESS method.
|
| |
|
| Submission date |
Jul 21, 2020 |
| Last update date |
Aug 28, 2022 |
| Contact name |
Ying-Tzu Huang |
| E-mail(s) |
d07447001@ntu.edu.tw
|
| Organization name |
National Taiwan University
|
| Department |
College of Medicine
|
| Street address |
No.1 Jen Ai road section 1
|
| City |
Taipei City |
| ZIP/Postal code |
100 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE154841 |
BLCAP knockdown HEK293 cells: shLuc vs. shBLCAP |
|
