|
| Status |
Public on Oct 01, 2017 |
| Title |
Senescent_rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
Senescent
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: U2OS
growth phase: Growth arrested
treatment: Dox treated
cell state: Senescent
|
| Treatment protocol |
For senescence, U2OS cells were treated with doxorubicin (1μM) for 2 h, then cells were recultured in fresh medium for 8 days.
|
| Growth protocol |
U2OS control cells were cultured in 10% FBS. For quiescence, U2OS cells were cultured in 0.1% serum for 96 h and then the G0 (quiescent) cells were sorted using FACS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Qiagen RNeasy mini kit following the manufacturer's recommendations. RNA was quantified using NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
The samples were labeled using Agilent Quick Amp Kit (Part number: 5190-0442). 500ng of total RNA was reverse transcribed using oligodT primer tagged to T7 promoter sequence. cDNA thus obtained was converted to double stranded cDNA in the same reaction. Further the cDNA was converted to cRNA in the in-vitro transcription step using T7 RNA polymerase enzyme and Cy3 dye was added into the reaction mix. During cRNA synthesis Cy3 dye was incorporated into the newly synthesized strands. cRNA obtained was cleaned up using Qiagen RNeasy columns(Qiagen, Cat No: 74106). Concentration and amount of dye incorporated was determined using Nanodrop. Samples that pass the QC for specific activity were taken for hybridization.
|
| |
|
| Hybridization protocol |
600ng of labeled cRNA were hybridized on the array using the Gene Expression Hybridization kit (Part Number 5190-0404; Agilent) in Sure hybridization Chambers (Agilent) at 65º C for 16 hours. Hybridized slides were washed using Agilent Gene Expression wash buffers (Part No: 5188-5327).
|
| Scan protocol |
Scanned on an Agilent scanner and Images were quantified using Agilent Feature Extraction Software
|
| Description |
Dox treated cells after culturing in fresh medium for 8 days
|
| Data processing |
Images were quantified using Feature Extraction Software (Version-10.7, Agilent). Feature extracted raw data was analyzed using GeneSpring GX software from Agilent. Normalization of the data was done in GeneSpring GX using the 75th percentile shift
|
| |
|
| Submission date |
Feb 12, 2017 |
| Last update date |
Oct 01, 2017 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL13252 |
| Series (1) |
| GSE94805 |
Gene expression profiling: Quiescence vs. Senescence |
|
