|
| Status |
Public on May 15, 2015 |
| Title |
LNCaP infected with shRNA library Replicate 2 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
LNCaP cells, shRNA library, bicalutamide
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: LNCaP
age: male
tissue: prostate
ethnicity: Caucasian
|
| Growth protocol |
LNCaP cells were infected with lentiviral shRNA library (Decode RNAi Viral Screening Library, Thermo Scientific Open Biosystems) and then treated with bicalutamide (1 micro M) or vehicle for one month.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from the transduced cells using the DNeasy Purification Kit (QIAGEN; Tokyo, Japan) according to the manufacturer’s protocol.
|
| Label |
Cy5
|
| Label protocol |
The integrated shRNAs prepared from LNCaP genomic DNAs were amplified using primers (Decode RNAi-GIPZ, annotated genes screening library-negative selection kit from Thermo Scientific) specific for the barcodes for the library plasmid DNA. The PCR products were gel-purified using the QIAquick PCR purification Kit (QIAGEN). Purified DNA fragments (1.5 micro g) from LNCaP cells treated with vehicle or bicalutamide were labeled with cyanine-3 (Cy3) or cyanine-5 (Cy5) dye, respectively, using the Genome DNA Enzymatic Labeling Kit (Agilent; Santa Clara, CA, USA) and purified by the removal of unbound cyanine dyes with an Ultracell YM-30 Microcon centrifugal filter device (Millipore Japan; Tokyo, Japan).
|
| |
|
| Channel 2 |
| Source name |
LNCaP cells, shRNA library, vehicle
|
| Organism |
Homo sapiens |
| Characteristics |
strain: LNCaP
age: male
tissue: prostate
ethnicity: Caucasian
|
| Growth protocol |
LNCaP cells were infected with lentiviral shRNA library (Decode RNAi Viral Screening Library, Thermo Scientific Open Biosystems) and then treated with bicalutamide (1 micro M) or vehicle for one month.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from the transduced cells using the DNeasy Purification Kit (QIAGEN; Tokyo, Japan) according to the manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
The integrated shRNAs prepared from LNCaP genomic DNAs were amplified using primers (Decode RNAi-GIPZ, annotated genes screening library-negative selection kit from Thermo Scientific) specific for the barcodes for the library plasmid DNA. The PCR products were gel-purified using the QIAquick PCR purification Kit (QIAGEN). Purified DNA fragments (1.5 micro g) from LNCaP cells treated with vehicle or bicalutamide were labeled with cyanine-3 (Cy3) or cyanine-5 (Cy5) dye, respectively, using the Genome DNA Enzymatic Labeling Kit (Agilent; Santa Clara, CA, USA) and purified by the removal of unbound cyanine dyes with an Ultracell YM-30 Microcon centrifugal filter device (Millipore Japan; Tokyo, Japan).
|
| |
|
| |
| Hybridization protocol |
Microarray hybridization was performed using the Oligo cDGH/ChIP-on-ChIP Hybridization Kit (Agilent).
|
| Scan protocol |
Scanned on an Agilent G2565BA scanner.
Images were quantified using Agilent Feature Extraction Software (version 8.1).
|
| Description |
Biological replicate 2 of 2.
|
| Data processing |
Agilent Feature Extraction Software (v 8.1) was used for background subtraction.
|
| |
|
| Submission date |
Aug 13, 2014 |
| Last update date |
May 15, 2015 |
| Contact name |
Kazuhiro Ikeda |
| Organization name |
Saitama Medical University
|
| Street address |
1397-1 Yamane
|
| City |
Hidaka |
| State/province |
Saitama |
| ZIP/Postal code |
350-1241 |
| Country |
Japan |
| |
|
| Platform ID |
GPL14820 |
| Series (1) |
| GSE60382 |
LNCaP cells: shRNA library with bicalutamide |
|
