Genome-wide DNA methylation sequencing of porcine brain
Genomic DNA from porcine brain tissue was extracted with Allprep DNA/RNA/Protein Mini Kit (Qiagen, Hilden, Germany) according to the manufacturers protocol. Then extracted DNA (1 g) was spiked with 1 ng of fragmented unmethylated phage  DNA (Promega, Madison, WI, USA, average size: 300 bp), which was end-repaired, A tailed, and adapter-ligated using Truseq DNA sample prep kit (Illumina Inc., USA) according to the manufacturers protocol. Adapter-ligated DNA was gel isolated and recovered using a QIAquick gel extraction kit (Qiagen, Hilden, Germany). After a cleanup with AMPure XP beads (Beckman Coulter Inc., Fullerton, CA, USA), bisulfite conversion was performed using EZ-DNA Methylation-Gold Kit (Zymo Research, Orange, CA, USA) according to the manufacturers protocol. Then, the bisulfite converted DNA was amplified with PfuTurbo Cx Hotstart DNA Polymerase (Agilent Technologies, Inc., Santa Clara, CA, USA) using the following thermal cycles: 95 C 5 min, 98 C 30 s, 12 cycles of (98 C 10 s, 65 C 30 s, 72 C 30 s), 72 C 5 min. Libraries were cleaned with AMpure XP beads and fragment length determined using an Agilent 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA). Quantified libraries (concentration > 2 nM) were then sequenced using the Illumina HiSeq2500 platform.