|
| Status |
Public on Sep 15, 2011 |
| Title |
H9 human embryonic stem cell replicate #1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
H9 hESCs
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H9 hESC
cell type: blastocytes
age: NA
|
| Growth protocol |
H9 hESCs were cultured with mouse feeder cells to promote self-renewal. H9 ESCs were also cultured in micropatterned chambers of 200μm2, 400μm2 and 800μm2 to control colony size, which previously has been shown induce differentiation along endoderm, mesoderm and neural-specified lineages, respectively (Lee et al., 2009; Peerani et al., 2007).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from ESCs using TRI reagent (Sigma-Aldrich) as per the manufacturer recommendations. PolyA+ mRNA was purified using Nucleotrap Midiprep kits (Clonetech). cDNA was synthesized using the WT-Ovation RNA Amplification System (Nugen) and was hybridized to custom AS microarrays as described previously (Pan et al., 2004).
|
| Label |
Cy3
|
| Label protocol |
cDNAs were labelled using the ULS labeling kit (Kreatech Biotechnology) according to manufacturer's recommendations.
|
| |
|
| Channel 2 |
| Source name |
H9 hESCs
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: H9 hESC
cell type: blastocytes
age: NA
|
| Growth protocol |
H9 hESCs were cultured with mouse feeder cells to promote self-renewal. H9 ESCs were also cultured in micropatterned chambers of 200μm2, 400μm2 and 800μm2 to control colony size, which previously has been shown induce differentiation along endoderm, mesoderm and neural-specified lineages, respectively (Lee et al., 2009; Peerani et al., 2007).
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted from ESCs using TRI reagent (Sigma-Aldrich) as per the manufacturer recommendations. PolyA+ mRNA was purified using Nucleotrap Midiprep kits (Clonetech). cDNA was synthesized using the WT-Ovation RNA Amplification System (Nugen) and was hybridized to custom AS microarrays as described previously (Pan et al., 2004).
|
| Label |
Cy5
|
| Label protocol |
cDNAs were labelled using the ULS labeling kit (Kreatech Biotechnology) according to manufacturer's recommendations.
|
| |
|
| |
| Hybridization protocol |
Microarrays were hybridized in duplicate with fluor reversal and washed using an HS 4800 hybridization station (Tecan)
|
| Scan protocol |
Scanned with an Agilent microarray scanner and images were processed by the Agilent Feature Extraction software (version 9.5)
|
| Data processing |
Variance stabilizing normalization (vsn) was performed using the vsn package within Bioconductor (version 2.1).
Raw, unprocessed input data are reported under the gVSNSignal and gVSNSignal columns of supplementary file.
Normalized intensity values from the microarray scans are reported under the gProcessedSignal and rProcessedSignal columns of supplementary file.
Normalized intensity values were input into the PATA algorithm (Mavadadi et al. In preparation) to generate final, processed data, i.e., estimates for percent exon inclusion levels (PATA output linked as supplementary file on Series record).
|
| |
|
| Submission date |
Sep 07, 2011 |
| Last update date |
Sep 15, 2011 |
| Contact name |
Xinchen Wang |
| E-mail(s) |
[email protected]
|
| Organization name |
MIT
|
| Street address |
32 Vassar St, D514
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL10691 |
| Series (2) |
| GSE30992 |
An Alternative Splicing Switch Regulates Embryonic Stem Cell Pluripotency and Reprogramming |
| GSE31948 |
An Alternative Splicing Switch Regulates Embryonic Stem Cell Pluripotency and Reprogramming [AS microarray] |
|
