|
| Status |
Public on Jan 06, 2012 |
| Title |
ESlncRNA over-expression vs. control erythroid progenitor cells rep1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
vector_induced_cells_rep1
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
developmental stage: E14.5
cell type: erythroid progenitor cells from fetal livers
overexpression: control
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from vector and ESlncRNA transduced erythroid progenitor cells (sorted as GFP positive cells), respectively. The quality of the RNA sample was determined by a 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy3
|
| Label protocol |
Cy-dye labeled cRNA samples were prepared using Agilent’s QuickAmp sample labeling kit. 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 |
ESIncRNA transduced erythoid progenitor cells rep1
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
developmental stage: E14.5
cell type: erythroid progenitor cells from fetal livers
overexpression: ESlncRNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from vector and ESlncRNA transduced erythroid progenitor cells (sorted as GFP positive cells), respectively. The quality of the RNA sample was determined by a 2100 Bioanalyzer (Agilent Technologies).
|
| Label |
Cy5
|
| Label protocol |
Cy-dye labeled cRNA samples were prepared using Agilent’s QuickAmp sample labeling kit. 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 8X66K expression arrays were hybridized according to our lab’s method, which differs slightly from the Agilent standard hybridization protocol. The hybridization cocktail consisted of cy-dye labeled cRNA, Agilent hybridization blocking components, and fragmentation buffer. The hybridization cocktails were fragmented at 60C 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 60C in an Agilent rotor oven set to maximum speed.
The arrays were treated with Wash Buffer #1 (6X SSPE / 0.005% n-laurylsarcosine) on a shaking platform at room temperature for 2 minutes, and then Wash Buffer #2 (0.06X SSPE) for 2 minutes at room temperature. 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 |
Data were analyzed using the limma package from Bioconductor. Loess method was used to make intensities consistent within each array, and aquantile method was applied to achieve consistency between arrays.
|
| |
|
| Submission date |
Jun 28, 2011 |
| Last update date |
Jan 06, 2012 |
| Contact name |
Wenqian Hu |
| E-mail(s) |
whu@wi.mit.edu
|
| Organization name |
Whitehead Institute
|
| Street address |
9 Cambridge Center
|
| City |
Cambridge |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
|
| Platform ID |
GPL10787 |
| Series (1) |
| GSE30279 |
ESlncRNA (AK148461) over-expression on gene expression |
|
