|
| Status |
Public on Jun 25, 2010 |
| Title |
WT vs KO differentiated NSCs, Replicate 2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
WT NSCs
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: wild type
age: Postnatal day 14-21
tissue: subventricular zones
cell type: neural stem cells
|
| Treatment protocol |
For spontaneous neuronal and glial differentiation, a cohort of undifferentiated NSCs, were re-plated on PO/FN substrate with mitogen for 2 days followed by withdrawal of mitogens for an additional 3 days.
|
| Growth protocol |
Postnatal day 14-21 day mouse lateral ventricle walls were dissected and mechanically dissociated. Single cell suspensions were plated at low density (10,000~20,000 cells cm-2) on cell culture dishes coated with poly-L-ornithine (PO)/fibronectin (FN), allowing individual cells to form spatially distinct colonies. Serial passage (less than 2 passages, or 2 weeks) allowed us to enrich undifferentiated NSCs to high purity (>95% Nestin+/Sox2+). The NSCs were maintained in serum-free B27 medium in presence of 10 ng/ml FGF-2/bFGF (Peprotech) and 10 ng/ml EGF (Invitrogen). The monolayer cultures were serially passaged every 4-7 days. To avoid the potential non-specific effect induced by culture adaption, only NSCs with limited in vitro culturing (less than 3 weeks) were used in the study.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA from WT and KO NSCs was reversed transcribed and labeled with Cy3- or Cy5-CTP using the Agilent Low RNA Input Fluorescent Linear Amplification Kit respectively.
|
| |
|
| Channel 2 |
| Source name |
KO NSCs
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Dnmt3a-null
age: Postnatal day 14-21
tissue: subventricular zones
cell type: neural stem cells
|
| Treatment protocol |
For spontaneous neuronal and glial differentiation, a cohort of undifferentiated NSCs, were re-plated on PO/FN substrate with mitogen for 2 days followed by withdrawal of mitogens for an additional 3 days.
|
| Growth protocol |
Postnatal day 14-21 day mouse lateral ventricle walls were dissected and mechanically dissociated. Single cell suspensions were plated at low density (10,000~20,000 cells cm-2) on cell culture dishes coated with poly-L-ornithine (PO)/fibronectin (FN), allowing individual cells to form spatially distinct colonies. Serial passage (less than 2 passages, or 2 weeks) allowed us to enrich undifferentiated NSCs to high purity (>95% Nestin+/Sox2+). The NSCs were maintained in serum-free B27 medium in presence of 10 ng/ml FGF-2/bFGF (Peprotech) and 10 ng/ml EGF (Invitrogen). The monolayer cultures were serially passaged every 4-7 days. To avoid the potential non-specific effect induced by culture adaption, only NSCs with limited in vitro culturing (less than 3 weeks) were used in the study.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Trizol following manufacturer's instructions
|
| Label |
Cy5
|
| Label protocol |
500 ng of total RNA from WT and KO NSCs was reversed transcribed and labeled with Cy3- or Cy5-CTP using the Agilent Low RNA Input Fluorescent Linear Amplification Kit respectively.
|
| |
|
| |
| Hybridization protocol |
825 ng Cy3- or Cy5-labelled cRNA were combined and hybridized to the Agilent 4x44K mouse whole genome microarray (G4122F) for 17 h at 65 ˚C (10 r.p.m.). Microarrays were washed according to Agilent's standard procedures.
|
| Scan protocol |
Microarry slides were scanned on at 5-micron resolution using an Agilent Technologies Scanner G2505B (US23502366).
|
| Description |
Biological replicate 2 of 2. Differentiated NSCs harvested after 3 days of mitogen withdrawal.
|
| Data processing |
Agilent Feature Extraction (FE) Software (v 9.5) was used for tiff image processing, background subtraction and Lowess normalization. Normalized signal intensity from FE was used for identifying differentially expressed transcripts between WT and KO NSCs in NIA array analysis tool (http://igsun.grc.nia.nih.gov/ANOVA).
|
| |
|
| Submission date |
Jun 21, 2010 |
| Last update date |
Jun 24, 2010 |
| Contact name |
Yi Sun |
| E-mail(s) |
ysun@mednet.ucla.edu
|
| Phone |
310-825-9506
|
| Organization name |
UCLA
|
| Department |
Molecular and Medical Pharmacology
|
| Street address |
635, Charles E. Young Dr. South
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (2) |
| GSE22473 |
Murine postnatal subventricular zone (SVZ) neural stem cells (NSCs): Wild-type (WT) vs. Dnmt3a-null (KO) |
| GSE22476 |
Dnmt3a-dependent non-promoter DNA methylation facilitates transcription of neurogenic genes |
|
