|
| Status |
Public on Nov 02, 2011 |
| Title |
SF126-tet-R175H-3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
SF126 human glioblastoma cell
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: SF126
genotype/variation: p53 R175H mutant
|
| Treatment protocol |
One million SF126-tet-R175H cells were seeded in 10-cm culture plates for 24 h. The medium was removed from the plates and the Decode RNAi Viral Screening Library (Thermo Scientific Open Biosystems, Huntsville, AL, USA) was added to the plate at the multiplicity of infection (MOI) of 0.3 with serum-free medium. After 6 h, the medium was replaced with virus-free medium. After 48 h, puromycin was added at a final concentration of 2 mg/ml to select the infected cells. Finally, 7 × 106 of lentivirus-infected SF126-tet-R175H cells were obtained. These cells theoretically contain 70,000 distinct shRNAs, and each cell should express a single shRNA product. These cells were divided into two groups, and each group was cultured with or without doxycycline for 10 days.
|
| Growth protocol |
The stable SF126 cell line expressing the doxycycline (Dox)-inducible p53 R175H mutant (SF126-tet-R175H) was constructed. Cells were cultured in RPMI1640 with 10% FBS at 37°C, 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from each group using the Blood & Cell Culture DNA Mini Kit (Qiagen), according to the manufacturer’s recommendation.
|
| Label |
Cy5
|
| Label protocol |
Barcode sequences corresponding to specific shRNAs were amplified by the following primers located outside the barcode sequence: forward, 5′-caaggggctactttaggagcaattatcttg-3′ reverse, 5′-ggttgattgttccagacgcgt-3′. Amplified PCR products were separated in 1.5% TAE agarose gel and extracted using Wizard SV Gel and PCR Clean-Up System (Promega, Madison WI, USA). Each purified PCR product was labeled with Cy5 (doxycycline-on group) or Cy3 (doxycycline-off group) using Agilent’s Genomic DNA Labeling Kit (Agilent Technologies, Inc., Santa Clara, CA, USA)
|
| |
|
| Channel 2 |
| Source name |
SF126 human glioblastoma cell
|
| Organism |
Homo sapiens |
| Characteristics |
cell name: SF126
genotype/variation: p53-null
|
| Treatment protocol |
One million SF126-tet-R175H cells were seeded in 10-cm culture plates for 24 h. The medium was removed from the plates and the Decode RNAi Viral Screening Library (Thermo Scientific Open Biosystems, Huntsville, AL, USA) was added to the plate at the multiplicity of infection (MOI) of 0.3 with serum-free medium. After 6 h, the medium was replaced with virus-free medium. After 48 h, puromycin was added at a final concentration of 2 mg/ml to select the infected cells. Finally, 7 × 106 of lentivirus-infected SF126-tet-R175H cells were obtained. These cells theoretically contain 70,000 distinct shRNAs, and each cell should express a single shRNA product. These cells were divided into two groups, and each group was cultured with or without doxycycline for 10 days.
|
| Growth protocol |
The stable SF126 cell line expressing the doxycycline (Dox)-inducible p53 R175H mutant (SF126-tet-R175H) was constructed. Cells were cultured in RPMI1640 with 10% FBS at 37°C, 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from each group using the Blood & Cell Culture DNA Mini Kit (Qiagen), according to the manufacturer’s recommendation.
|
| Label |
Cy3
|
| Label protocol |
Barcode sequences corresponding to specific shRNAs were amplified by the following primers located outside the barcode sequence: forward, 5′-caaggggctactttaggagcaattatcttg-3′ reverse, 5′-ggttgattgttccagacgcgt-3′. Amplified PCR products were separated in 1.5% TAE agarose gel and extracted using Wizard SV Gel and PCR Clean-Up System (Promega, Madison WI, USA). Each purified PCR product was labeled with Cy5 (doxycycline-on group) or Cy3 (doxycycline-off group) using Agilent’s Genomic DNA Labeling Kit (Agilent Technologies, Inc., Santa Clara, CA, USA)
|
| |
|
| |
| Hybridization protocol |
Barcode sequences labeled by Cy3 or Cy5 was hybridized on the barcode microarray in the hybridization oven (Agilent Technologies, Inc.) at 65°C for 17 h.
|
| Scan protocol |
After hybridization, the arrays were scanned with the Agilent DNA microarray scanner to quantify log2 Cy5/Cy3.
|
| Description |
Biological replicate 3 of 3. From the data (log2 cy5/ cy3), Change in the abundance of a particular shRNA barcode is tracked for 10 days by competitive hybridization between the Cy5 and Cy3 groups.
|
| Data processing |
Normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal according agilent software manufacturer's instructions.
|
| |
|
| Submission date |
Nov 01, 2011 |
| Last update date |
Nov 02, 2011 |
| Contact name |
imai hiroo |
| E-mail(s) |
imagen@idac.tohoku.ac.jp
|
| Phone |
+81-22-717-8543
|
| Fax |
+81-22-717-8547
|
| Organization name |
tohoku university
|
| Department |
clinical oncology
|
| Street address |
4-1 seiryo-machi
|
| City |
sendai |
| State/province |
Miyagi |
| ZIP/Postal code |
980-8575 |
| Country |
Japan |
| |
|
| Platform ID |
GPL14820 |
| Series (1) |
| GSE33362 |
Human glioblastoma cells: Tet-on(mutant p53 R175H expression) vs Tet-off(p53-null) |
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