|
| Status |
Public on Oct 01, 2014 |
| Title |
Notch_CFU_Gfi1KO-3 |
| Sample type |
RNA |
| |
|
| Source name |
Lin- BM
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
notch1 status: MigR1-Notch1IC-GFP
gfi1 ex4-5 status: KO
|
| Treatment protocol |
After 7 days, CFU were disrupted, counted and replated in 4-OHT (1uM) containing methylcellulose for an additional 7 days at 37 ºC in a humidified incubator with 5% CO2.
|
| Growth protocol |
Cells were flushed from tibias, femurs and iliac crests using PBS with 2% FBS and 1mM EDTA, lineage depleted using magnetic beads and put into StemSpan serum free media with cytokine support. Two days later, cells were transduced with MigR1-Notch1IC-GFP retroviral supernatants and FACS sorted 72 hours later. GFP+ cells were placed in methylcellulose for 7 days at 37 ºC in a humidified incubator with 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was prepared using the Qiagen RNeasy kit following the manufacturer's recommendations. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer Lab-On-A-ChIP Agilent 6000 Series II (Agilent Technologies, Santa Clara, CA).
|
| Label |
Cy3
|
| Label protocol |
Sample labeling and hybridization was performed according to the manufacturer's protocols using the One-Color Microarray-Based Gene Expression Analysis Manual (Low Input Quick Amp Version).
|
| |
|
| Hybridization protocol |
Samples were hybridized to the SurePrint G3 Mouse GE 8x60K Microarray (AMADID 028005).
|
| Scan protocol |
Microarray slides were hybridized overnight, washed and then scanned with Agilent G2505C Microarray Scanner.
|
| Description |
Gene expression after 1 week in 4-OHT containing methylcellulose
|
| Data processing |
The information about each probe on the array was extracted from the image data using Agilent Feature Extraction 10.10 (FE). This data is stored in the FE '.txt' files. Reading in the FE extraction files and extraction the median intensity values for each probe on the array. These intensities are not background corrected (this has been shown to only introduce noise), but are corrected for any scanner offset that was added to the original raw values. The dataset was filtered to remove positive control elements and any elements that have been flagged as outliers. Present (P), Marginal (M) or Absent (A) calls were made for each element on the array using in-house methodologies that take into account both the results of FE probe detection statistics and the intensities of the negative controls elements on the array. The median green (Cy3) intensities were normalized between the arrays using the Quantile Normalization package in 'R' (Bolstad et al., 2003).
|
| |
|
| Submission date |
Sep 26, 2012 |
| Last update date |
Oct 01, 2014 |
| Contact name |
H. Leighton Grimes |
| E-mail(s) |
Lee.Grimes@cchmc.org
|
| Phone |
513-636-6089
|
| Organization name |
Cincinnati Childrens Hospital Medical Center
|
| Department |
Immunobiology
|
| Lab |
Grimes
|
| Street address |
3333 Burnet Ave. MLC 7038
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL13912 |
| Series (1) |
| GSE41162 |
Lymphoid progenitor gene expression in Notch1 activated Gfi1-deleted cells |
|
