|
| Status |
Public on Oct 30, 2011 |
| Title |
5-hmC-mediated epigenetic dynamics (Mecp2.KO.2) [5hmC Capture and Seq] |
| Sample type |
SRA |
| |
|
| Source name |
Mecp2-/ytm1.1Bird Cerebellum
|
| Organism |
Mus musculus |
| Characteristics |
strain: Mecp2-/ytm1.1Bird
treatment: 5hmC enriched DNA
age: 6wk
Sex: Male
|
| Treatment protocol |
N/A
|
| Growth protocol |
N/A
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was purified from tissue using Wizard genomic DNA purification kit (Promega) with additional Proteinase K treatment and rehydrated in 10 mM Tris (pH 7.9). Genomic DNA samples were sonicated to 100-500 bp by Misonix sonicator 3000 (using microtip, 3 pulses of 30 seconds each with 2 min of rest and a power output level of two) or Bioruptor UCD-200 sonicator (Diagenode, Sparta). Output selector switch was set on High (H), and sonication interval was 30 seconds with 30 cycles of sonication performed. 5-hmC labelling reactions were performed in a 100-µL solution containing 50 mM HEPES buffer (pH 7.9), 25 mM MgCl2, 300 ng/μL sonicated genomic DNA (100-500 bp), 250 μM UDP-6-N3-Glu or UDP-Glu, and 2.25 μM wild-type BGT. The reactions were incubated for 1 hr at 37 °C. After the reaction, the DNA substrates were purified by Qiagen DNA purification kit or by phenol-chloroform precipitation and reconstituted in H2O. Biotin addition by click chemistry was performed by adding 150 μM dibenzocyclooctyne modified biotin into the DNA solution, and the reaction mixture was incubated for 2 hr at 37 °C. The DNA samples were then purified by Qiagen DNA purification kit. Biotin Labeled 5-hmC DNAs were purified/enriched by Pierce Monomeric Avidin Kit (Thermo) following manufacturerâs recommendations. After elution, the biotin-5-N3-gmC containing DNA was concentrated by 10 K Amicon Ultra-0.5 mL Centrifugal Filters (Millipore) and purified by Qiagen DNA purification kit. 25 ng genomic DNA, 5-hmC-captured DNA, or control captured DNA (in the absence of biotin) were used to initiate library preparation following the Illumina protocol for âPreparing Samples for ChIP Sequencing of DNAâ (Part# 111257047 Rev. A). DNA fragments ~150-300 bp were gel purified after the adapter ligation step and amplified by 18 cycles PCR.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Mecp2.KO.2
5-hydroxymethylcytosine-mediated epigenetic dynamics during neurodevelopment and aging
library strategy: 5hmC Capture and Sequencing
library selection5hmC Capture
|
| Data processing |
Processed data not provided.
|
| |
|
| Submission date |
Sep 12, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Keith E Szulwach |
| E-mail(s) |
[email protected]
|
| Phone |
404-712-0796
|
| Organization name |
Emory University
|
| Department |
Human Genetics
|
| Lab |
Jin Lab
|
| Street address |
615 Michael Street, Whitehead Suite 301
|
| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30322 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (2) |
| GSE32050 |
5-hydroxymethylcytosine-mediated epigenetic dynamics during neurodevelopment and aging [5hmC Capture and Seq] |
| GSE32188 |
5-hydroxymethylcytosine-mediated epigenetic dynamics during neurodevelopment and aging |
|
| Relations |
| SRA |
SRX097668 |
| BioSample |
SAMN00722046 |
