|
| Status |
Public on Feb 08, 2006 |
| Title |
Nanog RNAi replicate-3 |
| Sample type |
RNA |
| |
|
| Source name |
E14TG2a mouse embroyonic stem cells (ATCC, CRL-1841)
|
| Organism |
Mus musculus |
| Characteristics |
mouse embryonic stem cell
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Mouse embryonic stem cells were transfected with constructs expressing parental Nanog RNAi, using Lipofectamine 2000 (Invitorgen protocol). The cells were selected with 1ug/ml of Puromycin (Sigma) 24-hours after transfection for 3 days.
|
| Label |
biotin
|
| Label protocol |
Total RNAs were extracted directly from the cells using TRIzol reagent (Invitrogen). DNA contamination was removed by Dnase (Ambion) treatment and the RNA was further purified by Rneasy column (Qiagen Cat# 75144). Probes for hybridization were prepared by GeneChip One-Cycle target labeling kit (Affymetrix). Biotin-labeled cRNA was synthesizedusing the Enzo IVT labeling kit. 20 ug of the labeled cRNA was fragmented for target preparation in hybridization cocktail. Controls used in hybridization cocktail include Eukaryotic Hybridisation controls and Oligonucleotide B2 controls. SAPE (Streptavidin Phycoerythrin) and biotinylated antibody were used for microarray (Affymetrix Mouse Genome 430 2.0 Array) staining.
|
| |
|
| Hybridization protocol |
Prior to injection of hybridization cocktail, probes were primed by injection with hybridisation buffer (100mM MES, 1M [Na+], 20mM EDTA, 0.01% Tween-20 and placed in oven (45oC) for 10 minutes with rotation (60rpm). Hybridisation was performed by injection of the hybridization cocktail and incubating the probe in oven at 45oC with rotation (60rpm) for 16 hours. Probes were washed at the Fluidics FS-450 station with Buffer A (6X SSPE, 0.01% Tween 20) and B (100mM MES, 0.1M [Na+], 0.01% Tween-20). Staining of the probes was performed with 0.01mg/ml SAPE (Streptavidin Phycoerythrin) and biotinylated antibody (0.06mg/ml).
|
| Scan protocol |
standard Affymetrix procedures
|
| Description |
Nanog RNAi replicate-3
|
| Data processing |
RMA normalization
|
| |
|
| Submission date |
Feb 01, 2006 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Huck Hui Ng |
| E-mail(s) |
nghh@gis.a-star.edu.sg
|
| Phone |
+65 64788145
|
| Fax |
+65 64789004
|
| URL |
http://www.gis.a-star.edu.sg
|
| Organization name |
Genome Institute of Singapore
|
| Department |
Cell and Medical Biology
|
| Lab |
Cell and Medical Biology
|
| Street address |
60 Biopolis street, #02-01 Genome
|
| City |
Singapore |
| State/province |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE4189 |
The Oct4 and Nanog transcription network that regulates pluripotency in mouse embryonic stem cells |
|
| Relations |
| Reanalyzed by |
GSE119085 |
