|
| Status |
Public on Oct 13, 2010 |
| Title |
Wild type erythroblasts expressing ectopic Myc at supraphysiological levels 48hours 2 |
| Sample type |
RNA |
| |
|
| Source name |
E13.5 mouse fetal liver
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
treatment: Infection with MSCV-c-Myc-IRES-GFP encoding retroviruses at 0 hours in culture, followed by FACS sorting for highly GFP positive cells at 48 hours post infection
genotype/variation: Myc at supraphysiological levels
time in culture: 48 hours
|
| Treatment protocol |
Purified mouse fetal liver erythroblasts were cultured for 48 hours after infection with retroviruses encoding Myc or were cultured as uninfected cells for control.
|
| Growth protocol |
Fetal livers were isolated from E13.5 C57BL/6 mice embryos and mechanically dissociated by pipetting in PBS containing 20% fetal bovine serum (FBS). The dissociated cells were passed through 70 and 30 micron strainers to obtain single-cell suspensions. The cells were labeled with magnetic microbead-conjugated antibodies for TER119 and CD11b and passed through LD columns according to the manufacturer’s instructions for negative selection (Miltenyi Biotec). The purified TER119-negative, CD11b-negative erythroid progenitor cells were seeded in fibronectin coated plates and cultured in Iscove’s modified Dulbecco medium (IMDM) containing 15% FBS, 1% detoxified bovine serum albumin (StemCell Technologies), 200 μg/mL holo-transferrin (Sigma),10 μg/mL recombinant human insulin (Sigma), 2 mM L-glutamine, 10-4 M β-mercaptoethanol, and 2 U/mL erythropoietin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen, Germany) according to manufacturer instructions. RNA quality was assessed on the 2100 Bioanalyser (Agilent, USA) using the RNA 6000 Nano Chip kit (Agilent, USA) for intact 18S and 28S ribosomal peaks without significant degradation (RNA Integrity Number >8) for all samples.
|
| Label |
Biotin
|
| Label protocol |
500ng of total RNA from each sample was reverse transcribed into cDNA and in vitro transcribed into biotin-labelled cRNA using the Illumina TotalPrep RNA Amplification kit (Ambion, USA).
|
| |
|
| Hybridization protocol |
750ng of each cRNA sample was hybridized to MouseRef-8 v2.0 Expression BeadChip microarrays (Illumina, USA) as per manufacturer specifications.
|
| Scan protocol |
Microarrays were scanned on the BeadArray Reader (Illumina, USA) at scan factor 1 as per manufacturer specifications.
|
| Description |
Biological replicate 2
D2 Myc-GFPhigh2
|
| Data processing |
The microarray data was subjected to background subtraction and quantile normalization on the BeadStudio Data Analysis software (Illumina).
|
| |
|
| Submission date |
May 05, 2010 |
| Last update date |
Oct 13, 2010 |
| Contact name |
Senthil Raja Jayapal |
| E-mail(s) |
senthilraja@gis.a-star.edu.sg
|
| Phone |
+65 64788187
|
| Fax |
+65 6478-9005
|
| Organization name |
Genome Institute of singapore
|
| Department |
Stem cell and developmental biology
|
| Lab |
Stem cell group 4
|
| Street address |
#08-01, Genome, 60 Biopolis Street
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL6885 |
| Series (1) |
| GSE18558 |
Downregulation of Myc is essential for terminal erythroid maturation |
|
