DNA library preparation based on post-bisulfite adapter tagging

An adaptation of whole genome bisulfite sequencing that involves post-bisulfite adapter tagging (PBAT) was used to analyse the methylome of germ cells and early embryos at single-base resolution on a genome-wide scale, as previously performed [6].

With the exception of bovine oocytes, at least two replicate PBAT libraries were generated from each stage, each comprising the equivalents of ~ 80–150 cells per sample. In the case of the blastocyst stage, libraries were generated from single whole blastocysts. For porcine oocytes, PBAT was conducted on metaphase-II (MII)-stage oocytes obtained after in vitro maturation (IVM). Bovine oocytes were obtained by ovum pick-up after natural ovulation or hormonal stimulation and were processed for single-cell PBAT using the protocol described by [64] in this case, data from 28 individual oocyte libraries were merged for analysis.

For sperm methylome, bull and boar sperm DNA was extracted from ejaculates of two biological individuals. Cells were lysed in the lysis buffer containing 10% SDS with proteinase K, 20 mM Tris, 10 mM DTT, 10 mM EDTA, 150 mM NaCl and 10 mM KCl for 2 h at 50 C. Further, DNA was purified with phenol to chloroform at 1:1 ratio and precipitated with equal volume of isopropanol. DNA pellet was washed with 70% ethanol, air dried and dissolved in EB buffer. DNA concentration was measured using Nanodrop, and 10 ng was used for bisulfite conversion and PBAT library preparation as described below. For other biological timepoints, pools of oocytes (100), 2 cell embryos (40), 8 cell embryos (30), morulae (10) or individual blastocysts were lysed for 1 h in 1% SDS with proteinase K, and lysates were directly treated with bisulfite reagent using the Imprint DNA modification kit (Sigma, MOD50). DNA was eluted in EB buffer, and one round of first strand synthesis was performed using a biotinylated oligo 1 (5-[Btn] CTACACGACGCTCTTCCGATCTNNNNNNNNN-3). Samples were further treated with Exonuclease I, washed and eluted in 10 mM Tris-Cl and incubated with washed M-280 Streptavidin Dynabeads (Life Technologies) to pull down the biotinilated fraction of DNA. Second strand synthesis was performed using oligo 2 (5′-TGCTGAACCGCTCTTCCGATCTNNNNNNNNN-3′), and samples were amplified for 12 PCR cycles using indexed iPCRTag reverse primer [50] with KAPA HiFiHotStart DNA Polymerase (KAPA Biosystems) and purified using 0.8 × AgencourtAmpure XP beads (Beckman Coulter). Libraries were assessed for quality and quantity using high-sensitivity DNA chips on the Agilent Bioanalyzer. Two biological replicates were generated for each of biological timepoint and prepared for 100 bp single-end sequencing on Illumina HiSeq 1000 and sequenced at 4 samples per lane.