DNA methylation profiles (RRBS) of the muscle of pigs distinct in metabolic types and genetic background
DNA methylation profiles (RRBS) of the muscle of pigs distinct in metabolic types and genetic background
Longissimus muscle between 13th and 14th thoracic vertebrae was dissected immediately after slaughter. Tissue samples were flash frozen in liquid nitrogen and kept at -80 °C until DNA extraction. All experimental procedures including animal care and tissue sample collection were followed the guidelines for safeguarding and good scientific practice in accordance with the German Law of Animal Protection, officially authorized by the Animal Care Committee and authorities [Niedersächsischen Landesamt für Verbraucherschutz und Lebensmittelsicherheit (LAVES) 33.42502/01-47.05]. Genomic DNA was extracted from samples using a DNeasy kit (Qiagen) according to the manufacturer's recommendation including proteinase K digestion and RNase A treatment to obtain high purity DNA. 2 µg of DNA with a 1% spike-in control (unmethylated cl857 Sam7 Lambda DNA, Promega) was digested for 7 hours with 100U MspI (New England Biolabs) and then overnight with 50U TaqαI. The double-enzyme digested DNA fragments were end-repaired, A-tailed and ligated to TruSeq DNA adapters using a TruSeq Nano DNA library prep kit (Illumina). The adapter-ligated DNA fragments were size-selected to obtain inserts between 40 – 240bp. The DNA library was subjected to a bisulfite conversion using an EZ DNA Methylation Kit (Zymo Research). Library amplification was performed using a PfuTurbo Cx Hotstart DNA Polymerase kit (Stratagene). The quality of libraries was assessed on 2100 Bioanalyzer using a DNA chip (Agilent Technologies).