|
| Status |
Public on Apr 12, 2017 |
| Title |
SMAD4 |
| Sample type |
RNA |
| |
|
| Source name |
neonatal human epidermal keratintocyes
|
| Organism |
Homo sapiens |
| Characteristics |
transcription factor: SMAD4
|
| Treatment protocol |
Lipofectamine RNAi Max (Life Technologies) in OptiMEM medium was used for siRNA transfections. Pooled siRNA for each factor (Dharmacon) were used for knockdown in NHEK. 24 hours after knockdown, cells were induced to differentiate by the addition of high calcium media, and collected 24 hours later.
|
| Growth protocol |
Normal Human Epidermal Keratinocytes (NHEK) were purchased from LifeLine Technologies and grown according to the manufacturer’s instructions in DermaLife medium (LifeLine Tech) supplemented with DermaLife growth factors (LifeLine Tech). Cells were induced to differentiate by addition of high calcium medium (1.8mM Ca2+).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were collected and lysed in Trizol, followed purification with Zymo RNA extraction kit. RNA concentration and quality were quantified on a NanoDrop.
|
| Label |
Cy3
|
| Label protocol |
One-color labeling reactions were prepared using the Agilent One-Color Protocol (Version 5.7) with 300 ng total RNA input. RNA with the appropriate concentration of RNA spike-in controls is converted into double stranded cDNA using an olido (dT) primer linked to the T7 promoter sequence and MMLV-RT. The double stranded cDNA is then in vitro transcribed by T7 RNA polymerase, which simultaneously amplifies and incorporates cyanine 3-labeled CTP into the resulting cRNA. Labeled cRNA is then purified using Qiagen RNeasy columns and the labeling efficiency determined by the NanoDrop spectrophotometer.
|
| |
|
| Hybridization protocol |
The labeled cRNA is fragmented and placed on the human 8x15K microarray and hybridized at 65oC for ~17 hours.
|
| Scan protocol |
The arrays are washed and scanned at 5 μM resolution on an Agilent G2565CA High Resolution Scanner.
|
| Description |
253452510006_201107281010_S01 [2_2]
|
| Data processing |
Data was processed through Agilent’s Feature Extraction software version 10.7.31.
|
| |
|
| Submission date |
Aug 29, 2016 |
| Last update date |
Apr 13, 2017 |
| Contact name |
Bogi Andersen |
| Organization name |
University of California, Irvine
|
| Department |
Medicine
|
| Lab |
Andersen
|
| Street address |
839 Health Sciences Dr.
|
| City |
Irvine |
| State/province |
CA |
| ZIP/Postal code |
92697 |
| Country |
USA |
| |
|
| Platform ID |
GPL22382 |
| Series (1) |
| GSE86193 |
GRHL3 chromatin binding and the super-enhancer landscape are reorganized in different functional states of epidermal keratinocytes |
|
