|
| Status |
Public on Apr 12, 2016 |
| Title |
NT-shRNA treated primary kidney proximal tubular epithelial cells at day 6 replicate2 |
| Sample type |
RNA |
| |
|
| Source name |
PTECs_N2_NT_day 6
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: kidney
cell type: proximal tubular epithelial cells
|
| Treatment protocol |
Primary PTECs were transduced with SETD2 shRNA (sh1 and sh2), and non-targeting shRNA (NT); PTECs without virus treatment were included as a control. Both control and NT-PTECs (high MOI) were harvested at day 6 were named as PTECs_CON and PTECs_NT; PTECs harvested at day 16 were named as PTECs_S_CON and PTECs_S_NT; PTECs transduced with SETD2 shRNA (sh1 and sh2, low MOI) that were prevented from senescence were harvest at day 25 after transduction and named as PTECs_SH1/SH2.
|
| Growth protocol |
Primary proximal tubular epithelial cells (PTECs) were cultured in DMEM/F-12 GLUTMAX-1 (Sigma-Aldrich, St. Louis, MO, USA ) containing 10% FBS, 1% P/S (100 U/ml penicillin and 100 µg/ml streptomycin), 5μg/ml ITS (Sigma-Aldrich) and 5ng/ml EGF respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by using Gene JET RNA purification kit (Fermentas, Waltham, MA, USA) with the instruction offered by the manufacture.
|
| Label |
Cy5
|
| Label protocol |
First strand cDNA was synthesized from 50-100ng RNA, followed by cRNA amplification and labeling with Cy5 according to manufacturer's protocol (Agilent). Purification of Cy5 labeled cRNA was carried out with Qiagen RNeasy Mini kit.
|
| |
|
| Hybridization protocol |
Each Cy-5 sample was mixed with the same amount of a cy3-labeled sample, which was non-relevant for this study, and hybridized at 65°C overnight on Agilent-050524 SurePrint G3 Custom Human 8x60K Microarrays.
|
| Scan protocol |
On the following day, slides were washed and signals were scanned with GenePix 4000B (Agilent).
|
| Description |
mRNA expression analysis of NT-shRNA transduced PTECs at day 6 days
Sample 5
Cy3 signals in raw data files represent unrelated samples
|
| Data processing |
Signal intensities from scanned images were processed and converted into Linear and Lowess normalized data using Agilent Feature Extraction software version 10.7.3.1. Data was analyzed by GeneSpring GX 12.5 software (Agilent Technologies). The data was subject to quantile normalization without base line transformation.
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| |
|
| Submission date |
Apr 12, 2016 |
| Last update date |
Apr 12, 2016 |
| Contact name |
Jun Li |
| E-mail(s) |
nexusoooooo@gmail.com
|
| Phone |
+31641112670
|
| Organization name |
University Medical Center Groningen
|
| Department |
Genetics
|
| Lab |
Klaas Kok
|
| Street address |
Esdoornlaan
|
| City |
GRONINGEN |
| State/province |
Netherlands |
| ZIP/Postal code |
9741MG |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL18641 |
| Series (1) |
| GSE72792 |
Gene expression analysis of untreated and NT-shRNA transduced kidney proximal tubular epithelial cells (PTECs) at day 6 and day 16 after transduction, as well as SETD2-shRNA transduced PTECs at day 25 after transduction |
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