|
| Status |
Public on Feb 28, 2010 |
| Title |
Third shYY1 and shYY2 |
| Sample type |
RNA |
| |
|
| Source name |
HeLa cells transduced shYY1shYY2 control lentivirus.
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
|
| Treatment protocol |
Lentiviral pLKO-shRNAs were obtained from Sigma-Aldrich. The tracking numbers of the most effective shRNAs are TRCN0000019894 (YY1), TRCN0000016494 (YY2). To replace the puromycin-resistance gene in pLKO plasmids by neomycin-resistance for combined knockdown, an 827 bp fragment containing neoR gene was amplified from plasmid pCNH with primers 5'-TCA GGA TCC AGG ATC GTT TCG CAT GAT TGA AC-3' and 5'-TCA GGT ACC GAT GCA TGA GTC CCG CTC AGA AGA ACT CG-3', digested by Kpn I and BamH I, and ligated to Kpn I/BamH I-treated pLKO- shRNA plasmids. pLKO-shRNAs were packaged in 293T cells following the manufacturer’s standard protocol (Sigma-Aldrich). Spent medium of 293T cells was collected at 24-hour and 48-hour post-transfection, pooled, and cleared through a 0.45-micron syringe filter. To in-fect, HeLa cells were grown in 6-well culture plates in 2 ml medium and 0.5 ml of lenti-virus-containing medium was added. After 72 hours, cells were selected and maintained in medium containing 2 mg/ml of puromycin. To make combined shRNA transfectants, this process was repeated but selected using 500 mg/ml of Geneticin G418 for dual se-lections. Stable transfectants were split once a week and cells of less than fifteen pas-sages were used in all assays.
|
| Growth protocol |
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, and maintained in 37°C incubators with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared by Trizol Reagent (Invitrogen). Ten micrograms of RNA were used to hybridize the Affymetrix U133A2.0 human genome array.
|
| Label |
biotin
|
| Label protocol |
Affymetrix One-Cycle Target Labeling and Control Reagents (Part#900493) + Enzo BioArray High Yield RNA Transcript Labelling Kit (Part#42655)
|
| |
|
| Hybridization protocol |
Microarray = Affymetrix GeneChip Human Genome U133 Plus 2.0. Affymetrix Hybridization Oven= 645. Affymetrix Fluidics Station= 450. Fluidics Protocol= EukGE-WS2
|
| Scan protocol |
Affymetrix GeneChip Scanner= 3000-7G. Controller= GCOS
|
| Description |
n/a
|
| Data processing |
GCOS MAS 5
|
| |
|
| Submission date |
Feb 24, 2009 |
| Last update date |
Dec 22, 2009 |
| Contact name |
Emmett Vance Schmidt |
| E-mail(s) |
[email protected]
|
| Phone |
617-726-5707
|
| Fax |
617-726-8623
|
| Organization name |
Cancer Research Center at MGH
|
| Department |
Tumor Biology
|
| Lab |
Jackson 9 - Oncology Floor
|
| Street address |
55 Fruit St. - GRJ904
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE14964 |
Genome-wide analysis of YY2 versus YY1 target genes |
|
