|
| Status |
Public on Apr 02, 2010 |
| Title |
MeDIP-Seq_RJF-Liver_YG |
| Sample type |
SRA |
| |
|
| Source name |
liver
|
| Organism |
Gallus gallus |
| Characteristics |
breed: red junglefowl
medip antibody: anti-5-methycytosine mouse mAb
|
| Treatment protocol |
Two 7-day-old chickens were utilized in this experiment, one red jungle fowl and one avian broiler. The chickens were fed with the same diet and sacrificed according to local standards of animal welfare issues. Liver and muscle tissues taken from each animal were flash frozen in liquid nitrogen and then stored at -80°C.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was isolated by phenol-chloroform extraction. DNA was sonicated to small segments and DNA fragments ranging from 200-350bp were retracted by gel excision with gel extraction kit (Qiagen,). The recovered DNA was first 5’ and 3’ end blunting, phosphorylating and repairing by T4 Polynucleotide Kinase and T4 DNA Polymerase (NEB). After addition of an ATP in the 3’ end, an Illumina sequencing primer adapter was ligated to the DNA using the Quick LigationTM Kit (Qiagen). DNA was recovered by MinElute® PCR Purification Kit (Qiagen) and used for meDIP. Our meDIP method was modified from previous study. For each sample, we incubated 4μg denatured DNA with 32μg anti-5-methylcytosine mouse monoclonal antibody in 400μl IP buffer (10mM Tris-HCl, pH7.5, 280mM NaCl, 1mM EDTA) at 4°C for 5.5 hr. Then we added 100μl Dynabeads Protein G and Protein A (Dynal) to the mix and incubated at 4℃ for 5.5 hr. The following step was the same as in the method described by Xiaoyu Zhang. After meDIP, the DNA was divided into three fractions: the unbound, washed and bound fractions. With a high methylated region as a positive control verified by bisulfite sequencing and a region without CpG dinucleotides as a negative control, we carried out realtime-PCR to determine the relative enrichment of the control region in the above three fractions. DNA in the bound fraction from qualifying meDIP experiment was PCR amplified with sequencing primers provided by Illimina using PhusionTM High-Fidelity PCR Master Mix (Finnzymes) under the following conditions: 3min at 98°C; followed by 18 cycles of 98°C for 15s, 65°C for 30s, 72°C for 20s; and a final extension for 5min. PCR products were recovered using MinElute PCR Purification Kit (Qiagen) and used for Solexa sequencing.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
meDIP against methylated DNA
|
| Data processing |
The reads generated during a Illumina/Solexa Sequencing run are 35 bp in length. The resulting FASTQ files were aligned to the chicken reference genome (galGal3) by the open-source aligner the Mapping and Assembly with Qualities (MAQ).
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/)
|
| |
|
| Submission date |
Apr 01, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
qinghe li |
| E-mail(s) |
lqhcau@163.com
|
| Organization name |
China Agricultural university
|
| Street address |
no2, yuanmingyuan west road
|
| City |
beijing |
| ZIP/Postal code |
100193 |
| Country |
China |
| |
|
| Platform ID |
GPL10223 |
| Series (2) |
| GSE21167 |
Examination of whole genome DNA methylation status of two chicken breeds |
| GSE21170 |
Genome-wide Differential DNA Methylation in the Wild and Domestic Chicken |
|
| Relations |
| SRA |
SRX018733 |
| BioSample |
SAMN00010878 |
