title: Illumina Genome Analyzer IIx paired end sequencing; Methylome of 9 canine transmissible veneral tumour (CTVT) samples
design: Methylome of 9 canine transmissible veneral tumour (CTVT) samples
library_construction_protocol: Biopsies of were collected from dogs undergoing diagnostic assessment at the Department of Surgery and Obstetrics and Gynaecology, Faculty of Veterinary Medicine, University of Messina, Italy.  All the procedures on animals treated in Italy, including housing, conformed to EU regulations of animal welfare and are approved by the Italian Ministry of Health being in agreement with the directive D.P.C.M. of 28th February 2003. Qiagen AllPrep Micro kit according to the manufacturer's protocol Sonication with the Diagenode Bioruptor. Following fragmentation (180-230 bp fragments), library preparation consists of DNA end repair, 3′-dA tailing and adapter ligation. DNA was incubated with 150 ng anti-5-methylcytidine at 4°C for 15 hrs using automation and manufacturer reagents and protocols (Diagenode). Following immunoprecipitation, DNA was incubated with 1 μg proteinase K (Diagenode) at 55°C for 15 mins then 95°C for 15 mins. Each sample was subject to quality control using quantitative PCR (qPCR) with primers designed to amplify methylated/unmethylated control DNA (see https://www.ncbi.nlm.nih.gov/pubmed/22402632 for details). After confirming enrichment of methylated DNA in the immunoprecipitated fraction, DNA was purified using AmpureXT magnetic beads and amplified by 12 cycles adapter-mediated PCR. PCR products were purified using Ampure magnetic beads and run out on 2% low-melting point agarose gel. PCR fragments were excised and purified (QIAGEN). The resulting libraries (300–350 bp) were quality controlled using an Agilent 2100 Bioanalyzer with DNA1000 chips.
attributes.tag: Experimental Factor: Stationary
attributes.value: tumor grading
attributes.tag: Experimental Factor: CTVT17
attributes.value: individual