|
| Status |
Public on Aug 16, 2016 |
| Title |
CSC_humanGC1_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Cancer stem cell, induced from human gastric cancer tissue of patient GC1, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Cancer stem cell, induced from human gastric cancer tissue of patient GC1
gender: Male
age: 67
|
| Treatment protocol |
The isolated clones were subcultured in each well of gelatin-coated 24-well plates. After an expansion culture, each clone was further cultured to each well of gelatin-coated 6-well plates and finally cultured in a gelatin-coated 100-mm dish. The expanded clones were treated with a dissociation solution (0.25% trypsin–EDTA [Gibco] and 1% collagenase [Invitrogen]) or 0.25% trypsin–EDTA and passaged in mTeSR1 supplemented with 10–20 μM Y-27632 (Calbiochem and Wako) to avoid cell death. Those clones were cultured with the MEFs (5 X 10^4 cells/cm^2) mainly in mTeSR1 medium and occasionally in Primate ESC medium (ReproCell) on gelatin-coated dishes. Total RNA was prepared from each 100-mm dish and subjected to microarray analysis.
|
| Growth protocol |
The cancer tissue-derived cells were seeded on collagen-coated dishes with DMEM supplemented with 10% fetal bovine serum. One day later, the cells at approximately 5-10% confluency were incubated with the pantropic retrovirus vector solution (OCT3/4, KLF4, and SOX2) at 37°C for one day. The pantropic retrovirus vector solution was prepared. Mitomycin C-treated mouse embryonic fibroblasts were seeded following the infection. The culture was replaced with confluency. The confluent culture was further refreshed with mTeSR1 medium every day from day 15. Each clone was isolated.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Using the AllPrep DNA/RNA Mini kit (Qiagen), total RNA was prepared from each clone that was cultured on the MEFs (5 X 10^4 cells/cm^2) with mTeSR1 (StemCell Technologies) medium in gelatin-coated 100-mm dishes before long-term serial passage. RNA was quantified using a NanoDrop ND-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 μg RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA, USA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 μg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 250 ml of 2× Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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 with 37°C GE Wash buffer 2 (Agilent), then dried immediately by brief centrifugation.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B) using one color scan setting for 1x44k array slides (Scan Area 61×21.6 mm, Scan resolution 10um, Dye channel is set to Green and Green PMT is set to 100%)
|
| Description |
Gene expression of induced cancer stem cells established from human gastric cancer tissue-derived cells
iPS-GC1-2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v5_95_Feb07 and Grid: 014850_D_20070820) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Normalized signal intensity by Agilp in series supplementary file.
|
| |
|
| Submission date |
Jun 29, 2016 |
| Last update date |
Aug 16, 2016 |
| Contact name |
Akimasa Seno |
| Organization name |
Okayama University
|
| Department |
Applied Chemistry and Biotechnology
|
| Lab |
Nano-Biotechnology
|
| Street address |
1-1, Tsushima-Naka, 1-chome, Kita-Ku
|
| City |
Okayama |
| State/province |
Okayama |
| ZIP/Postal code |
7008530 |
| Country |
Japan |
| |
|
| Platform ID |
GPL6480 |
| Series (2) |
| GSE83879 |
Gene expression of developed cancer stem cells [iPS-GC1] |
| GSE83883 |
Gene expression of developed cancer stem cells |
|
