title: NextSeq 500 sequencing; Genome-wide DNA methylation reprogramming in porcine primordial germ cells
design: Genome-wide DNA methylation reprogramming in porcine primordial germ cells
library_construction_protocol: 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).