NextSeq 500 sequencing; Genome-wide DNA methylation reprogramming in porcine primordial germ cells
Gonads were extracted from fetuses at the corresponding embryonic day. Gonads were dissected and primordial germ cells (PGCs) were labelled with anti-SSEA1 microbeads, then separated on MS columns in a Magnetic-activated cell sorting (MACS) separation unit (MiltenyiBiotec Inc., UK) following the manufacturer's instructions.  Cells were collected in 10 µl of PBS and lysed in 15 µl lysis buffer containing 0.5% sodium dodecyl sulphate (SDS), 1 µl proteinase K and EB, using the Imprint DNA modification kit (Sigma). The bisulphite converted DNA was eluted with elution buffer (EB) and one round of first-strand synthesis performed using a biotinylated oligo 1 (5-[Btn]CTACACGACGC-TCTTCCGATCTNNNNNNNNN-3). Samples were further treated with Exonuclease I, washed and eluted with EB and subjected to solid-phase reversible immobilization (SPRI) purification and incubated with 20 µl Spretavidin Dynabeads to capture the biotinylated fraction of DNA. Second strand synthesis was performed using oligo 2 (5'-TGCTGAACCGCTCTTCCGATCTNNNNNNNNN -3') and samples were amplified for 15 PCR cycles using indexed iPCRTag reverse primers with KAPA HiFi HotStart DNA polymerase (Kapa Biosystems) and purified using SPRI beads (Agencourt Ampure XP bead).