|
| Status |
Public on May 16, 2012 |
| Title |
RF_RAD_rep3, MeDIP |
| Sample type |
SRA |
| |
|
| Source name |
Rongchang pig, retroperitoneal adipose
|
| Organism |
Sus scrofa |
| Characteristics |
breed: Rongchang
gender: female
tissue: adipose: retroadipose: peritoneal adipose
medip antibody: anti-methylcytidine monoclonal
antibody vendor: Diagenode
|
| Growth protocol |
Three pig breeds with distinguished fat levels were analyzed: Landrace (a leaner, Western breed), Rongchang (a fatty, Chinese breed) and Tibetan (a feral, indigenous, Chinese breed that has not undergone artificial selection).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
We randomly selected three female and three male pigs from each breed as biological replicates. We obtained ten tissues for each individual for a total of 180 samples, and sequenced each sample separately. Each MeDIP library was subjected to paired-end sequencing using Illumina HiSeq 2000 and a 50 bp read length. Five ug original DNA was isolated using the E.Z.N.A. HP Tissue DNA Midi Kit (OMEGA) and was sonicated to ~100-500 bp fragments with a Bioruptor sonicator (Diagenode). Then, libraries were constructed using the Illumina Paired-End protocol consisting of end repair, 'A' base addition and adaptor ligation steps, and were performed using Illumina's Paired-End DNA Sample Prep kit following the manufacturer's instructions. Adaptor-ligated DNA was immunoprecipitated using the monoclonal anti-methylcytidine antibody in the Magnetic Methylated DNA Immunoprecipitation Kit (Diagenode). The specificity of the enrichment was confirmed by qPCR using SYBR green mastermix (Applied Biosystems) and primers for positive and negative internal control DNA of non-human samples supplied in the Magnetic Methylated DNA Immunoprecipitation Kit (Diagenode). Cycling of qPCR validation was 95C 5 min, followed by 40 cycles 95C 5 s and 60C 1 min. The enriched fragments with methylation and 10% input DNA were purified on ZYMO DNA Clean & Concentrator-5 columns following the manufacturer's instructions. DNA was eluted in 30 ul buffer EB and its concentration was measured. Enriched fragments were amplified by adaptor-mediated PCR in a final reaction volume of 50 ul consisting of 23 ul purified DNA, 25 ul Phusion DNA polymerase mix (NEB) and 2ul PCR primers. Amplification consisted of 94C 30 s, 10 cycles of 94C 30s, 60C 30 s, 72C 30 s then prolong with 5 min at 72C and products were held at 4C. Amplifications quality and quantity were evaluated by Agilent 2100 Analyzer DNA 1000 chips purified by 2% agarose gel and eluted in 15 ul buffer EB. Ultra-high-throughput 50 bp pair-end sequencing was carried out using the Illumina HiSeq 2000 according to manufacturer's instructions. Raw sequencing data were processed by the Illumina base-calling pipeline.
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
MeDIP library for the paired-end sequencing is 50 bp read length.
|
| Data processing |
After filtering the low-quality reads, the MeDIP-seq data were aligned to the UCSC pig reference genome (Sscrofa9.2) using SOAP2 (Version 2.21). The differentially methylated regions (DMRs) were identified using our newly developed method by calculating variation of single CpGs. Refer to Methods of the related manuscript for details.
|
| |
|
| Submission date |
Jul 02, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Mingzhou Li |
| E-mail(s) |
mingzhou.li@163.com
|
| Phone |
86-835-2886000
|
| Organization name |
Sichuan Agricultural University
|
| Department |
College of Animal Science and Technology
|
| Lab |
Institute of Animal Genetics and Breeding
|
| Street address |
No.46 Xinkang Road
|
| City |
Ya'an |
| State/province |
Sichuan |
| ZIP/Postal code |
625014 |
| Country |
China |
| |
|
| Platform ID |
GPL11429 |
| Series (1) |
| GSE30344 |
An atlas of DNA methylomes in pig adipose and muscle tissues |
|
| Relations |
| SRA |
SRX082755 |
| BioSample |
SAMN00651660 |
