|
| Status |
Public on Mar 01, 2017 |
| Title |
AK4-WT |
| Sample type |
RNA |
| |
|
| Source name |
EB after two day RA induction
|
| Organism |
Mus musculus |
| Characteristics |
tissue: embryoid bodies
gender: male
|
| Treatment protocol |
To get the time series samples from ES cell to motor neurons, RA/Shh were applied from day 2 to day 5 during the differention process.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA isolation by Trizol, DNase treated by TURBO DNA-free Kit (Life Technology), and checked with Bioanalyzer 2100 (Agilent) for RIN > 9.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (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 |
825 ng of Cy3-labelled cRNA (specific activity >6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Mouse GE 8x60K Microarray (G4858A-028005) 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 (G2505C) using one color scan setting for 8x60k array slides.
|
| Description |
Gene expression in wt EB after RA treated two days
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software10.5.1 (Agilent) using default parameters (protocol GE1-105_D08 and Grid: 028005_D_F_20110711) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Dec 09, 2016 |
| Last update date |
Mar 02, 2017 |
| Contact name |
Jun-An Chen |
| E-mail(s) |
jachen@imb.sinica.edu.tw
|
| Organization name |
Academia Sinica
|
| Department |
Institute of Molecular Biology
|
| Lab |
N517
|
| Street address |
128 Academia Road, Section 2
|
| City |
Taipei |
| ZIP/Postal code |
11529 |
| Country |
Taiwan |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE91080 |
Analysis of gene expression along the Hoxa cluster at five time-points during motor neuron differentiation |
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