|
| Status |
Public on Dec 24, 2009 |
| Title |
HES3 EB medium 7day rep2 |
| Sample type |
RNA |
| |
|
| Source name |
human embryonic stem cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HES3
culture condition: EB
culture day: 7
|
| Extracted molecule |
total RNA |
| Extraction protocol |
All the EBs harvested at the various time points were rinsed twice with PBS followed by extraction of total RNA using RNeasy® Plus Micro Kit (Cat. No- 74034). The eluate volume was 12 ul. 1.5 ul of the eluted sample was run on Nanodrop™ 1000 (Thermo Fisher Scientific) to determine the yield. 50 ng of the sample was used to run on Agilent Bionalyzer 2100 with the Agilent RNA 6000 Nanochip (Cat No. 5067-1511) using the default settings to determine the integrity of the labeled sample.
|
| Label |
biotin
|
| Label protocol |
The Illumina® TotalPrep RNA Amplification Kit (Cat No. AMIL1791) was used for amplification of total RNA for microarray analysis. Briefly, 100 ng of total RNA was used for first strand reverse transcriptase (RT) reaction with T7 oligo (dT) primer which was carried out for 2 hours at 42oC, followed by a second strand cDNA synthesis at 16oC for 2 hours. Amplification of biotinylated cRNA was carried out by in vitro transcription with biotin UTP using purified the cDNA products at 37oC for 16 hours. 1.5 ul of the sample was run on Nanodrop to determine the concentration of the purified labeled cRNA. 100 ng of the sample was used to run on Agilent Bionalyzer 2100 with the Agilent RNA 6000 Nanochip (Cat No. 5067-1511) using the default settings to determine the integrity of the labeled sample.
|
| |
|
| Hybridization protocol |
1.5 µg of biotinylated cRNA was used for each array in the Human WG-6 v3.0 BeadChip (Illumina). The BeadChips were hybridized at 58oC for 16 hours and washed according to the standard Illumina protocol.
|
| Scan protocol |
The arrays were scanned with the Illumina Beadstation 500GX using Scan Factor 1.0 and PMT 501.
|
| Description |
replicate 2
|
| Data processing |
Scanned data were retrieved using BeadStudio Gene Expression Module v3.4 and inspected with the quality control parameters. Data was background corrected using the default settings prior to further processing. The background corrected raw data was pre-processed with Bioconductor/R using the ‘lumi’ package (Du et al. 2008. Bioinformatics 24, 1547-1548). The data was transformed using the VST method (Lin et al 2008. Nucleic Acids Res. 36, e11), quantile normalized and filtered with detection p-value <0.01. Quality control of the data was carried out before and after normalization with density plots, signal intensity boxplots and pairwise plots implemented with the ‘lumi’ package.
|
| |
|
| Submission date |
Jun 08, 2009 |
| Last update date |
Dec 24, 2009 |
| Contact name |
Woon-Khiong Chan |
| E-mail(s) |
dbscwk@nus.edu.sg, g0500139@nus.edu.sg
|
| Organization name |
National University of Singapore
|
| Department |
Biological Sciences
|
| Lab |
Molecular Genetics Laboratory
|
| Street address |
S2-05-19, 14 Science Drive 4
|
| City |
Singapore |
| ZIP/Postal code |
117543 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL6884 |
| Series (1) |
| GSE16484 |
Development of a three dimensional in vitro teratoma assay for pluripotent human embryonic stem cells |
|
