|
| Status |
Public on Oct 15, 2013 |
| Title |
Cont 0h rep2 |
| Sample type |
RNA |
| |
|
| Source name |
FDCPmix_Cont 0h
|
| Organism |
Mus musculus |
| Characteristics |
cell line: FDCPmix
infected with: Empty vector
|
| Treatment protocol |
GATA1ERT and Pu.1ERT were subcloned into the pHR-SIN-CSGWEmGFP lentiviral expression construct under control of the SFFV promoter. Gata2 and Pu.1 shRNAs were subcloned into Lentilox 3.7. Recombinant plasmids were packaged into lentiviral particles esssentially by published procedures. FDCPmix cells were transduced at an MOI of between 50 and 200. For gene expression analysis, samples of FDCPmix cells in self-renewal conditions were transduced with lentiviral particles encoding GATA1ERT or PU.1ERT fusion proteins linked to ires-GFP, with empty virus used as a control. GFP+ cells were sorted after 3 days and expanded for a further 7 days under standard conditions, before re-sorting on GFP and addition of 2M 4OH-tamoxifen. Cells were harvested after 0h and 24h of induction and total RNA analyzed by microarray.
|
| Growth protocol |
FDCPmix were grown in 20% Fischer's medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolate using TRIzol reagent, and RNA concentration and integrity were determined using RNA 6000 Nano RNA kit (Agilent) on BioAnalyzer 2100 (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Total RNA (150ng) and Agilent One-Color RNA Spike-In were reverse-transcribed and linearly amplified in the presence of Cy3-labeled CTP using Low RNA Input One-Color Kit (Agilent) following Agilent protocols, to generate Cy3-labeled cRNA for hybridisation to high-density microarrays. Quality and yield/specific activity of cRNA were determined using RNA 6000 Nano kit and spectrophotometer (NanoDrop ND- 1000). All samples generated comparable quality cRNA profiles, with specific activities in the range of 10.3 to 12.2 pmol Cy3/mg cRNA.
|
| |
|
| Hybridization protocol |
Each Cy3-labeled cRNA sample (1.6ug as per NanoDrop reading) was hybridised to an individual 44K subarray of 4x44K high-density microarray slide (Whole Mouse Gene Expression Microarrays, Agilent), washed, scanned and feature-extracted as per Agilent protocols.
|
| Scan protocol |
Scanning and feature extraction was performed as per Agilent protocols
|
| Description |
ER_2
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software (Agilent v10.1.1.1) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Aug 19, 2013 |
| Last update date |
Oct 15, 2013 |
| Contact name |
Shamit Soneji |
| E-mail(s) |
shamit.soneji@med.lu.se
|
| Organization name |
BMC
|
| Street address |
Sölvegatan 19
|
| City |
Lund |
| ZIP/Postal code |
221 84 |
| Country |
Sweden |
| |
|
| Platform ID |
GPL7202 |
| Series (2) |
| GSE49990 |
Dynamic analysis of gene expression and genome wide transcription factor binding during lineage-specification of multipotent progenitors [ER] |
| GSE49991 |
Dynamic analysis of gene expression and genome wide transcription factor binding during lineage-specification of multipotent progenitors |
|
