|
| Status |
Public on Dec 21, 2015 |
| Title |
Ischemic muscle DNA from type 2 diabetic mice |
| Sample type |
SRA |
| |
|
| Source name |
Gastrocnemius muscle
|
| Organism |
Mus musculus |
| Characteristics |
strain: IGF-II/LDLR-/-ApoB100/100 , with C57BL/6J background
tissue: Gastrocnemius muscle
|
| Treatment protocol |
Mice were anesthetized by subcutaneous injection of mixture containing Rompun® (10 mg/kg) (Xylazine, Orion pharma, Espoo, Finland) and Ketamine (100 mg/kg) (Ketalar, Pfizer Oy, Espoo, Finland) before surgery. Unilateral hindlimb ischemia was induced by ligating femoral artery proximal to the bifurcation of superficial and deep femoral artery as described previously.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tissue samples were snap frozen in liquid nitrogen at day 7 after ischemia induction and later DNA was isolated using DNeasy Tissue kit (Qiagen).
The quality and quantity of DNA was analyzed with nanodrop. 5 µg of genomic DNA was sheared into small fragments with a mean size of 150 bp by using a Covaris™ S2 sonicator System (www.covaris.com). Quality of the fragmentation was analyzed with Bioanalyzer (www.agilent.com). Fragmented DNA samples with sufficient yield and an appropriate size distribution were used for preparation of DNA fragment libraries. Methylated DNA was captured according to the manufacturer’s protocol [The Methyl Miner™ methylated DNA Enrichment Kit (Invitrogen)]. Methylated DNA was isolated from fragmented whole genomic DNA (5 ng –25 μg) via binding to the methyl-CpG binding domain of human MBD2 protein, which is coupled to paramagnetic Dynabeads RM-280 Streptavidin via a biotin linker. The amount of input DNA was 3 ug. The captured DNA fragments were eluted with 2000 mM NaCl. End Polishing Enzyme 1 and End Polishing Enzyme 2 were used to convert DNA that has damaged or incompatible 5'-protruding and/or 3'-protruding ends to 5'-phosphorylated, blunt-ended DNA. End Polishing Enzyme 1 and ATP were also included for phosphorylation of the 5'-ends of the blunt-ended DNA to allow for subsequent adaptor ligation. End repaired samples were purified with the PureLink™ PCR Purification columns (Invitrogen). The double-stranded P1 and P2 adaptors were hybridized and ligated to the ends of fragmented DNA. These adaptors contained defined sequences required for SOLiD sequencing. For multiplexing, the SOLiD™ Fragment Library Barcoding Kit Module 1–16 was used. The samples were nick translated and subsequently amplified using Library PCR Primers 1 and 2 and Platinum® PCR Amplification Mix. After amplification, PCR samples were purified with the Agencourt AM XP beads. The amplified and purified DNA was run on a 1 % agarose gel. The correct sized ligation products (150 to 300 bp) were cut from the gel according to the 50 bp ladder (Invitrogen). The size-selected product was elute extracted (QIAquick gel extraction kit, Qiagen) from the gel. The quality and size of the samples were then analyzed with Bioanalyzer. Libraries were quantitated with Qubit fluorometer (Invitrogen) and quantitative PCR to warrant the accuracy. SOLiD Library TaqMan Quantitation Kit was used for determining the molar concentration of amplified template in a SOLiD library. The uniformity of fragment size of libraries was confirmed with Bioanalyzer. For SOLiD fragment libraries the target size of the fragments was 150-300 bp. Templated bead preparation was performed by emulsion PCR (ePCR), which involves monoclonal amplification of individual species of template DNA from a complex library pool. During ePCR, multiple copies of a single DNA sequence were coated onto a single 1-μm magnetic bead (SOLiD™ P1 DNA Beads) within a water-in-oil emulsion droplet. SOLiD™ EZ Bead™ instrumentation was used for templated bead preparation. Before ePCR, equal molar amounts (250 pM) of each of the bar coded libraries were mixed together and one ePCR reaction was performed with the library mixture (template 1 pM). After ePCR and enrichment of the template beads, 800 mlj beads were deposited onto one single chamber sequencing glass slide. Subsequently, samples were sequenced on the SOLiD4 next generation sequencing instrument (Applied Biosystems) on one flow cell with SOLiD4 chemistry for 50 bp reads.
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
AB SOLiD 4 System |
| |
|
| Data processing |
Color space data were preprocessed and aligned to the reference genome using Life Technologies' LifeScope software
Relative methylation scores were calculated with MEDIPS package in R/Bioconductor
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using MEDIPS package in R/Bioconductor. Scores represent the relative methylation values.
|
| |
|
| Submission date |
Nov 06, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Mohan Babu |
| E-mail(s) |
babu.mohan@uef.fi
|
| Phone |
+358403553685
|
| Organization name |
A.I.Virtanen Institute, University of Eastern Finland
|
| Department |
Biotechnology and Molecular Medicine
|
| Lab |
Molecular Medicine
|
| Street address |
Neulaniementie 2
|
| City |
Kuopio |
| ZIP/Postal code |
70211 |
| Country |
Finland |
| |
|
| Platform ID |
GPL14602 |
| Series (2) |
| GSE42092 |
Genome wide DNA methylation sequencing of ischemic skeletal muscle from normal, hyperlipidemic and type 2 diabetic mice |
| GSE65803 |
Differential promoter methylation of macrophage genes correlates with impaired vascular growth in ischemic muscles of hyperlipidemic and type 2 diabetic mice |
|
| Relations |
| SRA |
SRX203063 |
| BioSample |
SAMN01801809 |
