|
| Status |
Public on Dec 31, 2013 |
| Title |
HDF_SAHA-PIP5_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Human Dermal Fibroblasts, SAHA-PIP5(SAHA-PIP E), replicate 1
|
| Organism |
Homo sapiens |
| Characteristics |
cell source: human dermal fibroblasts derived from normal human breast skin
gender: female
age: 54y
treatment: 48hr treatment with SAHA-PIP5 (SAHA-PIP E)
|
| Treatment protocol |
HDFs within the passage P6 were trypsinized for 5 min at 37 °C, and were resuspended in the fresh medium to a concentration of 1.5 x 10^5 cells/2ml in a 35 mm plate and were grown for 24 hours. The medium was then removed and replaced with 2 ml of fresh medium supplimented with DMSO solusion of SAHA, SAHA-PIPs 1-32 (final concentration: 1 μM). The cultures were incubated for 48 hours.
|
| Growth protocol |
54-year-old female Caucasian HDFs were maintained in Dulbecco's modified eagle medium (DMEM, Life Technologies) containing 10% fetal bovine serum (FBS, Japan Serum).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNeasy mini kit (Qiagen, Valencia, CA) following the manufacturer's recommendations. RNA was quantified using a Nanodrop ND1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labelled cRNA was prepared from 100 ng RNA using the Low Input Quick Amp labeling kit, one-color (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600ng of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes using 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE Microarray kit 8x60 Ver2.0 (G4851B) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute at 37°C with GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent Scanner (G2539A) using one color scan setting for AgilentG3 GX array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression in HDFs after 48hr treatment with SAHA-PIP5 (SAHA-PIP E)
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.10.1.1 (Agilent) using default parameters (protocol: GE1_1010_Sep10 and Grid: 039494_D_F_20120411) to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Dec 13, 2013 |
| Last update date |
Dec 31, 2013 |
| Contact name |
Junichi Taniguchi |
| E-mail(s) |
[email protected]
|
| Organization name |
Kyoto University
|
| Department |
Department of Chemistry, Graduate School of Science
|
| Lab |
Sugiyama Laboratry
|
| Street address |
Kitashirakawa-Oiwakecho, Sakyo-Ku
|
| City |
Kyoto |
| ZIP/Postal code |
606-8502 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17077 |
| Series (2) |
| GSE53317 |
Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts (Agilent 1-color) |
| GSE53319 |
Distinct DNA-based epigenetic switches trigger transcriptional activation of silent genes in human dermal fibroblasts |
|
