|
| Status |
Public on Jun 25, 2010 |
| Title |
WTvsKO NSCs, H3K27me3 ChIP-chip (1/4) |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
WT NSC, immunoprecipitated DNA by H327me3 antibodies
|
| Organism |
Mus musculus |
| Characteristics |
tissue: postnatal SVZ
genotype/variation: wild type
cell type: neural stem cells
experimental design: IP/IP
|
| Growth protocol |
postnatal SVZ NSCs were maintained in undifferentiated states in presence of mitogens (10 ng/ml EGF and 10 ng/ml bFGF)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Each ChIP-chip assay was performed with about 20 million cells following Agilent mammalian ChIP-on-chip protocol (March, 2006). Briefly, cells were cross-linked with formaldehyde for 10 min at room temperature. Sonicated chromatin was immunoprecipitated overnight at 4 C with 10 ug immobilized (on 100 ul Dynal protein-G beads, Invitrogen) antibodies to Dnmt3a (Santa Cruz, sc-20703) or H3K27me3 (Millipore, 07-449). MeDIP-chip was perfromed as previously described with minor modifications (Weber et al., 2005). Briefly, 5 ug sonicated, heat-denatured genomic DNA was immunoprecipitated with 5 ul of monoclonal antibody against 5-methylcytidine (Eurogentec) at 4 C overnight. Immunoprecipitated methylated DNA was collected with 30 ul Dynal protein-G beads for 2 h at 4 C.
|
| Label |
Cy3
|
| Label protocol |
Whole cell extract and immunoprecipitated DNA were amplified using the Whole Genome Amplification Kit (Sigma), and 5 ug of amplified ChIP/MeDIP or input samples were labeled (5’ Cy5- or Cy3-random nonamers, TriLink Biotechnologies) using the standard protocol (NimbleGen Arrays User’s Guide for ChIP-chip analysis).
|
| |
|
| Channel 2 |
| Source name |
KO NSC, immunoprecipitated DNA by H327me3 antibodies
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Dnmt3a-null
cell type: neural stem cells
tissue: postnatal SVZ
experimental design: IP/IP
|
| Growth protocol |
postnatal SVZ NSCs were maintained in undifferentiated states in presence of mitogens (10 ng/ml EGF and 10 ng/ml bFGF)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Each ChIP-chip assay was performed with about 20 million cells following Agilent mammalian ChIP-on-chip protocol (March, 2006). Briefly, cells were cross-linked with formaldehyde for 10 min at room temperature. Sonicated chromatin was immunoprecipitated overnight at 4 C with 10 ug immobilized (on 100 ul Dynal protein-G beads, Invitrogen) antibodies to Dnmt3a (Santa Cruz, sc-20703) or H3K27me3 (Millipore, 07-449). MeDIP-chip was perfromed as previously described with minor modifications (Weber et al., 2005). Briefly, 5 ug sonicated, heat-denatured genomic DNA was immunoprecipitated with 5 ul of monoclonal antibody against 5-methylcytidine (Eurogentec) at 4 C overnight. Immunoprecipitated methylated DNA was collected with 30 ul Dynal protein-G beads for 2 h at 4 C.
|
| Label |
Cy5
|
| Label protocol |
Whole cell extract and immunoprecipitated DNA were amplified using the Whole Genome Amplification Kit (Sigma), and 5 ug of amplified ChIP/MeDIP or input samples were labeled (5’ Cy5- or Cy3-random nonamers, TriLink Biotechnologies) using the standard protocol (NimbleGen Arrays User’s Guide for ChIP-chip analysis).
|
| |
|
| |
| Hybridization protocol |
34 ug of Cy5- and Cy3-labeled samples were combined and hybridized to each mouse whole genome tiling microarray (Roche/NimbleGen 4-array set) for 16-20 h at 42 ˚C using NimbleGen hybridization System 4.
|
| Scan protocol |
Microarrays were scanned using the Agilent scanner at 5 micron resolution.
|
| Data processing |
Data were extracted and analyzed using NimbleScan v2.5 (Roche/NimbleGen). Dnmt3a binding sites were identified by a sliding window-base statistical algorithm in NimbleScan v2.5 (FDR<1% or <5%). For identifying probes associated with a significant increase in H3K27me3 levels or a significant decrease in DNA methylation level in KO NSCs as compared to WT NSCs, a non-parametric one-sided Kolmogorov-Smirno (KS) test was used (KS score>2).
Processed data VALUE definition: Log2 (ch2_signal / ch1_signal)
|
| |
|
| 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 |
GPL7523 |
| Series (2) |
| GSE22475 |
Genome-wide location analysis of Dnmt3a-mediated epigenetic regulation in murine postnatal subventricular zone (SVZ) neural stem cells (NSCs) [NimbleGen] |
| GSE22476 |
Dnmt3a-dependent non-promoter DNA methylation facilitates transcription of neurogenic genes |
|
