|
| Status |
Public on Sep 19, 2011 |
| Title |
Trophoblast rep3 |
| Sample type |
RNA |
| |
|
| Source name |
H9 human ES cells treated with BMP-4
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: BMP-4 6 days
|
| Treatment protocol |
H9 cells, grown on matrigel were treated with BMP-4 for 6 days
|
| Growth protocol |
H9 human embryonic stem cells were grown in standard culture conditions to maintain them in an undifferentiated state. Colonies were transferred to matrigel for 2 passages before treatment
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from undifferentiated and BMP-4 treated H9 cells using a Qiagen Rneasy kit.
|
| Label |
Cy5
|
| Label protocol |
Sample amplification and labeling procedures were carried out by using Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies). Total RNA was reverse transcribed into first strand and second strand cDNA by MMLV-RT using an oligo dT primer that incorporates a T7 promoter sequence. cDNA is then used as a template for in vitro transcription in the presence of T7 RNA polymerase and Cyanine labeled CTPs. The labeled cRNA is purified using RNeasy micro kit (Qiagen) and followed by quantification on both concentrations of cRNA and dye labeled. RNA spike-in controls (Agilent Technologies) are added to RNA samples before amplification and labeling according to manufacturer’s protocol. Entire amount of each samples labeled with Cy3 or Cy5 are mixed with control targets (Agilent Technologies). Fragmentation is carried out by incubating at 60C for 30 minutes and stopped by adding equal volume of 2x GE Hi-RPM hybridization buffer (Agilent Technologies).
|
| |
|
| Hybridization protocol |
Fragmented targets are added onto a microarray, assembled into a hybridization chamber (Agilent Technologies) and hybridized at 60ºC for 17 hours in a hybridization oven with rotation. Hybridized microarrays are washed and dried according to the Agilent microarray processing protocol.
|
| Scan protocol |
Microarrays were scanned using an Agilent G2505B Scanner controlled by Agilent Scan Control 7.0 software. Data were extracted with Agilent Feature Extraction 9.1 software.
|
| Description |
H9 human ES cells treated with BMP-4
|
| Data processing |
gProcessedSignals from all six samples were quantile normalized using R. The data matrix contains the normalized values.
|
| |
|
| Submission date |
Sep 07, 2011 |
| Last update date |
Sep 19, 2011 |
| Contact name |
Hongkai Ji |
| E-mail(s) |
hji@jhsph.edu
|
| Organization name |
Johns Hopkins Bloomberg School of Public Health
|
| Department |
Department of Biostatistics
|
| Street address |
615 North Wolfe Street, Rm E3638
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
| |
|
| Platform ID |
GPL4133 |
| Series (2) |
| GSE31965 |
Gene expression comparison between undifferentiated H9 cells and trophoblasts |
| GSE32220 |
Cell-type independent MYC target genes reveal a primordial signature involved in biomass accumulation |
|
