|
| Status |
Public on Dec 31, 2012 |
| Title |
5hmC NPC 3/4 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
5hmC enriched DNA fraction after pull-down with 5hmC antibodies. DNA was isolated from NPC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: neural progenitor cells, cortex
age: E15.5
enrichment method: hMeDIP
antibody: 5hmC
|
| Treatment protocol |
no treatment
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For MIRA, sonicated DNA was incubated with MBD2b-GST and MBD3L1-His overnight at +4C. Methylated DNA was captured by using magnetic GST beads, purified and amplified by using LM-PCR. For ChIp-ChIp experiments, cells were fixed with 1% formaldehyde in PBS1x for 10 minutes at RT and briefly quest by adding of 0.125M Glycine. Sonicated chromatin was incubated with appropriate antibodies overnight and captured by A/G agarose beads. Purified decrosslinked DNA was amplified by LM-PCR procedure. For hMeDIP, linker ligated DNA was denatured and incubated with 5hmC antibodies overnight at +4C with further purification with A/G plus beads and genome amplification by LM-PCR. For 5hmC enrichment with Hydroxymethyl Collector (Active Motif), sonicated DNA was glycosylated, labeled with biotin and purified according manufacturer instructions with further genome amplification by LM-PCR.
|
| Label |
Cy5
|
| Label protocol |
According to standard NimbleGen Protocol
|
| |
|
| Channel 2 |
| Source name |
Input DNA from NPC
|
| Organism |
Mus musculus |
| Characteristics |
cell type: neural progenitor cells, cortex
age: E15.5
enrichment method: none, Input
antibody: none, Input
|
| Treatment protocol |
no treatment
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For MIRA, sonicated DNA was incubated with MBD2b-GST and MBD3L1-His overnight at +4C. Methylated DNA was captured by using magnetic GST beads, purified and amplified by using LM-PCR. For ChIp-ChIp experiments, cells were fixed with 1% formaldehyde in PBS1x for 10 minutes at RT and briefly quest by adding of 0.125M Glycine. Sonicated chromatin was incubated with appropriate antibodies overnight and captured by A/G agarose beads. Purified decrosslinked DNA was amplified by LM-PCR procedure. For hMeDIP, linker ligated DNA was denatured and incubated with 5hmC antibodies overnight at +4C with further purification with A/G plus beads and genome amplification by LM-PCR. For 5hmC enrichment with Hydroxymethyl Collector (Active Motif), sonicated DNA was glycosylated, labeled with biotin and purified according manufacturer instructions with further genome amplification by LM-PCR.
|
| Label |
Cy3
|
| Label protocol |
According to standard NimbleGen Protocol
|
| |
|
| |
| Hybridization protocol |
According to NimbleGen Kit
|
| Scan protocol |
Follow NimbleScan's default using Agilent Scanner
|
| Description |
Cells were isolated by FACs sorting from nmouse cortex using GFP/RFP reported system at 15.5E embryonic development. GFP+ (under Nestin promoter), RFP - (Under Dbx promoter)
|
| Data processing |
Cy5/Cy3 log2 ratio scaled by NimbleScan software v2.5 with Tukey Biweight mean
|
| |
|
| Submission date |
May 22, 2012 |
| Last update date |
Dec 31, 2012 |
| Contact name |
Xiwei Wu |
| E-mail(s) |
xwu@coh.org
|
| Organization name |
City of Hope National Medical Center
|
| Department |
Computational and Quantitative Medicine
|
| Street address |
1500 E. Duarte Rd.
|
| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
| |
|
| Platform ID |
GPL15590 |
| Series (1) |
| GSE38118 |
Dynamics of 5-hydroxymethylcytosine and chromatin marks in mammalian neurogenesis |
|
