|
| Status |
Public on Aug 31, 2012 |
| Title |
PARN KD C2C12_actinomycinD_0_min_rep_3 |
| Sample type |
RNA |
| |
|
| Source name |
PARN KD C2C12 with actinomycin D, time 0 min, rep 3
|
| Organism |
Mus musculus |
| Characteristics |
cell line: C2C12 myoblast cell line
genotype/variation: PARN Knock Down
agent: actinomycin D
time after actinomycin d treatment: 0 min
|
| Treatment protocol |
To inhibit transcription, cells were treated with actinomycin D (8µg/ml) for 30 min prior to the start of the time course.
|
| Growth protocol |
The mouse C2C12 myoblast cell line was obtained from the American Type Culture Collection (CRL1772). Two derivatives of the C2C12 cell line, CTRL and PARN KD, were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1µg/ml puromycin, 10U/ml penicillin and 10µg/ml streptomycin in 5% CO2 at 37°C. Transfections were performed using Lipofectamine 2000 and transfection efficiency was routinely in the 50-70% range. The CTRL cell line stably transfected with LKO1 vector was described previously (GSE21236). The PARN KD clonal cell line was generated by puromycin selection following transduction with an shRNA-encoding lentivirus derived from the LKO1 vector.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected in Trizol™ (Invitrogen) at the indicated time points following the incubation and total RNA was prepared according to the manufacturers instructions. RNA concentration, purity, and quality were determined via Bioanalyzer (Agilent).
|
| Label |
biotin
|
| Label protocol |
Biotinylated DNA probes were prepared according to Whole Transcript Sense Target Labeling Manual (Affymetrix)
|
| |
|
| Hybridization protocol |
300ng of total RNA for half life arrays were used to generate labeled cDNA fragments for hybridization to Mouse Affymetrix Gene 1.0 ST Arrays following the manufacturer’s protocol (GeneChipTM WT cDNA Synthesis Kit #900652 and #900720).
|
| Scan protocol |
GeneChips were scanned using Affymetrix GeneChip 3000G
|
| Description |
This is sample was collected at 0 min after actinomycin D treatment
PARNKD.0.C.CEL
|
| Data processing |
All the probe sets were applied median normalization by Affymetrix Power Tools (APT) with ‘no adjustment’ option and were normalized to the 5th percentile value of all probe sets on the same array.
|
| |
|
| Submission date |
Feb 21, 2012 |
| Last update date |
Aug 31, 2012 |
| Contact name |
Ju Youn Lee |
| Organization name |
UMDNJ-New Jersey Medical School
|
| Department |
Biochemistry
|
| Street address |
185 South Orange Ave. MSB-E501
|
| City |
Newark |
| State/province |
NJ |
| ZIP/Postal code |
07101 |
| Country |
USA |
| |
|
| Platform ID |
GPL6246 |
| Series (1) |
| GSE35944 |
The PARN deadenylase targets a discrete set of mRNAs for decay and regulates cell motility in mouse myoblasts |
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