|
| Status |
Public on Nov 04, 2015 |
| Title |
gArp6 KO 2days |
| Sample type |
RNA |
| |
|
| Source name |
gArp6 KO 2days
|
| Organism |
Gallus gallus |
| Characteristics |
conditinoal knockout chicken DT40 cell for gArp6
|
| Treatment protocol |
Targeted disruption constructs for the ARP6 gene were generated so that genetic fragments encoding exons 6-11 were replaced with a histidinol- (hisD) or puromycin- (puro) resistance cassette under the control of the beta-actin promoter. DT40 cells were cultured at 38˚C in Dulbecco’s modified medium supplemented with 10% fetal calf serum, 1% chicken serum, penicillin and streptomycin. The target constructs were transfected with a Gene Pulser II electroporator (Bio-Rad, Tokyo, Japan). Histidinol (Sigma) at a final concentration of 1 mg/ml, zeocin (Invitrogen) at a final concentration of 1 mg/ ml, and puromycin (Clontech) at a final concentration of 0.5 ug/ml were used to select stable transfectants.
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| Growth protocol |
The chicken DT40 cells and conditional knockout cells for Arp6 with DT40 cell were cultured at 38˚C in Dulbecco’s modified medium supplemented with 10% fetal calf serum, 1% chicken serum, penicillin and streptomycin. To suppress expression of the tetracycline - responsive Arp6 transgene, tetracycline (Sigma) was added to the culture medium to a final concentration of 2 ug/ml.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
The total RNA was extracted with RNeasy Maxi (QIAGEN) according to the manufacturer protocol. The poly-A mRNA was isolated from total RNA using Oligotex-dT30 Super (JSR corporation).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA was synthesized according to Affymetrix GeneChip Expression Analysis Technical Manual (Affymetrix)Shortly, 2 ug of poly-A mRNA was reverse transcribed to cDNA using 100 pmol of a T7-oligo (dT) primer (Invitrogen), and biotinylated cRNA was prepared by using the cDNA as template.
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| |
|
| Hybridization protocol |
For hybridization, 15 ug of biotinylated cRNA was fragmented and added into a hybridization cocktail containing four biotinylated hybridization controls (bioB, bioC, bioD, and cre) as recommended by the manufacturer. GeneChip Chicken genome Arrays (Affymetrix) were hybridized with the cocktail at 45˚C for 16 hours.
|
| Scan protocol |
After washing with Non-Stringent wash buffer (6xSSPE, 0.01% Tween20) and staining with SAPE in the Affymetrix GeneChip Fluidics Station 400, the GeneChips were read using the Affymetrix GeneChip scanner 3000 7G.
|
| Description |
To suppress expression of gArp6, conditional konckout cells for gArp6 were cultured with tetracycline to a final concentration of 2 ug/ml for 2 days.
|
| Data processing |
These systems and data analyses were operated with the GeneChip Operating Software 1.3.
|
| |
|
| Submission date |
Nov 12, 2008 |
| Last update date |
Nov 04, 2015 |
| Contact name |
Yukako Oma |
| E-mail(s) |
labharata@gmail.com
|
| Organization name |
Tohoku University
|
| Department |
Graduate School of Agricultural Science
|
| Lab |
Molecular Biology
|
| Street address |
Tsutsumidori-Amamiyamachi 1-1, Aoba-ku
|
| City |
Sendai |
| ZIP/Postal code |
981-8555 |
| Country |
Japan |
| |
|
| Platform ID |
GPL3213 |
| Series (1) |
| GSE14220 |
The nuclear actin-related protein Arp6 contributes to the radial positioning of chromosome territories. |
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