|
| Status |
Public on Aug 01, 2012 |
| Title |
UtxKO +/- RA |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
UtxKO ESC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: ES cells
genotype/variation: Utx KO
developmental stage: V6.5
treatment: none
|
| Treatment protocol |
ES media was replaced with ES media minus LIF plus 1x10-7M retinoic acid (RA). Cells were cultured for an additional 48 hours with one media exchange.
|
| Growth protocol |
ES cells were grown on gelatin in an undifferentiated state (ES medium (+LIF)).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed with PBS and RNA was extracted using RNAeasy Kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
Cy-dye labeled cRNA samples were prepared using Agilent’s Low Input Quick Amp Sample Labeling Kit. Input was 200ng total RNA. Briefly, first and second strand cDNA are generated using MMLV-RT enzyme and an oligo-dT based primer. In vitro transcription is performed using T7 RNA polymerase and either cyanine 3-CTP or cyanine 5-CTP, creating a direct incorporation of dye into the cRNA.
|
| |
|
| Channel 2 |
| Source name |
UtxKO ESC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: ES cells
genotype/variation: Utx KO
developmental stage: V6.5
treatment: RA for 48 hrs
|
| Treatment protocol |
ES media was replaced with ES media minus LIF plus 1x10-7M retinoic acid (RA). Cells were cultured for an additional 48 hours with one media exchange.
|
| Growth protocol |
ES cells were grown on gelatin in an undifferentiated state (ES medium (+LIF)).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were washed with PBS and RNA was extracted using RNAeasy Kit (Qiagen).
|
| Label |
Cy5
|
| Label protocol |
Cy-dye labeled cRNA samples were prepared using Agilent’s Low Input Quick Amp Sample Labeling Kit. Input was 200ng total RNA. Briefly, first and second strand cDNA are generated using MMLV-RT enzyme and an oligo-dT based primer. In vitro transcription is performed using T7 RNA polymerase and either cyanine 3-CTP or cyanine 5-CTP, creating a direct incorporation of dye into the cRNA.
|
| |
|
| |
| Hybridization protocol |
Agilent (mouse 4x44k) expression arrays were hybridized according to the Agilent standard hybridization protocol. The hybridization cocktail consisted of 825 ng cy-dye labeled cRNA for each sample, Agilent hybridization blocking components, and fragmentation buffer. The hybridization cocktails were fragmented at 60°C for 30 minutes, and then Agilent 2X hybridization buffer was added to the cocktail prior to application to the array. The arrays were then hybridized for 16 hours at 65°C in an Agilent rotor oven set to maximum speed. The arrays were treated with Agilent Wash Buffer 1 at room temperature for 2 minutes, and then AgilentWash Buffer 2 for 2 minutes at 37°C. The arrays were then dipped briefly in acetonitrile before a final 30 second wash in Agilent Wash 3 Stabilization and Drying Solution, in the hood using a stir plate and stir bar at room temperature
|
| Scan protocol |
Arrays were scanned using an Agilent scanner G2565BA and the data was extracted using Agilent’s Feature Extraction software v10.1.1.1 set to the default two-color gene expression protocol.
|
| Data processing |
Within-array LOESS and Across-array quantile normalization using limma for R
|
| |
|
| Submission date |
Jul 10, 2012 |
| Last update date |
Aug 01, 2012 |
| Contact name |
Albert W Cheng |
| E-mail(s) |
awcheng@mit.edu
|
| Organization name |
Whitehead Institute
|
| Street address |
Room 453, 9 Cambridge Center
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL10333 |
| Series (2) |
| GSE39222 |
The X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex specific manner |
| GSE39473 |
The X-linked H3K27me3 demethylase Utx is required for embryonic development in a sex specific manner [Agilent array data] |
|
