|
| Status |
Public on Jun 08, 2013 |
| Title |
MIRA |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
HES-2_MIRA enriched DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: embryonic stem cell line HES-2
sample type: MIRA enriched sample
|
| Growth protocol |
Matrigel (BD)/mTesR1 (STEMCELL technologies) standard protocol
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
HES-2 (ES02) human embryonic stem cells were adapted from mitotically inactive MEF feeders to matrigel substrate (BD Biosciences) and mTesR1 medium (STEMCELL Technologiesaccording to manufacturer's protocol. Adaptation was for 4 passages and the final passage number was p72. DNA was extracted from Tra 1-60+ cells isolated by flow cytometry and prepared for MIRA-chip analysis. 500 ng of sonicated and fragmented DNA was enriched for methylated DNA using the MethylCollector Ultra Kit (Active Motif). Eluted DNA was purified with MinElute reaction cleanup kit (Qiagen). MIRA-enriched and 10 ng of sonicated input DNA was subsequently amplified using the Whole Genome Amplification kit2 (Sigma).
|
| Label |
Cy5
|
| Label protocol |
1 µg methylation DNA was directly labeled by Klenow (Roche NimbleGen) random priming with Cy3 nonamers per manufacturer's protocol (http://www.nimblegen.com/).
|
| |
|
| Channel 2 |
| Source name |
HES-2_INPUT DNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: input DNA
cell line: embryonic stem cell line HES-2
|
| Growth protocol |
Matrigel (BD)/mTesR1 (STEMCELL technologies) standard protocol
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
HES-2 (ES02) human embryonic stem cells were adapted from mitotically inactive MEF feeders to matrigel substrate (BD Biosciences) and mTesR1 medium (STEMCELL Technologiesaccording to manufacturer's protocol. Adaptation was for 4 passages and the final passage number was p72. DNA was extracted from Tra 1-60+ cells isolated by flow cytometry and prepared for MIRA-chip analysis. 500 ng of sonicated and fragmented DNA was enriched for methylated DNA using the MethylCollector Ultra Kit (Active Motif). Eluted DNA was purified with MinElute reaction cleanup kit (Qiagen). MIRA-enriched and 10 ng of sonicated input DNA was subsequently amplified using the Whole Genome Amplification kit2 (Sigma).
|
| Label |
Cy3
|
| Label protocol |
1 µg methylation DNA was directly labeled by Klenow (Roche NimbleGen) random priming with Cy3 nonamers per manufacturer's protocol (http://www.nimblegen.com/).
|
| |
|
| |
| Hybridization protocol |
The labeled DNA was precipitated with 0.1 volume 5M NaCl and 1 volume isopropanol, and hybridized in 45 ul of buffer containing 20% formamide, 1.2 M betaine, 0.1 ug/ul herring sperm DNA and 10 ug of human COT1 DNA (Invitrogen). Arrays were hybridized in Maui hybridization stations for 16-18 h at 42C, and then washed in Wash I, Wash II, Wash III according to manufacturer's protocols (http://www.nimblegen.com/)
|
| Scan protocol |
Arrays were scanned on an Roche NimbleGen MS200 scanner per manufacturer's protocol (http://www.nimblegen.com/).
|
| Description |
embryonic stem cell line
|
| Data processing |
NimbleScan 2.6.0.0
|
| |
|
| Submission date |
Dec 04, 2012 |
| Last update date |
Oct 16, 2018 |
| Contact name |
Charles David Warden |
| Organization name |
City of Hope
|
| Department |
Department of Molecular and Cellular Biology
|
| Street address |
1500 East Duarte Rd
|
| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
| |
|
| Platform ID |
GPL16353 |
| Series (2) |
| GSE42310 |
COHCAP: City of Hope CpG Island Analysis Pipeline |
| GSE42734 |
COHCAP MIRA / 450k Comparison [MIRA] |
|
