4.3. WGBS Library Preparation and Sequencing

One microgram of genomic DNA was fragmented in 50 µL of 0.5× Tris-EDTA buffer using Bioruptor Plus sonication device (Diagenode, Liège, Belgium) under 10 cycles of 30 s ON and 90 s OFF power conditions. Then, fragmented DNA was purified with 1.8× Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA) and eluted in 40 µL of ddH₂O. Whole genome bisulfite sequencing libraries were prepared using the NEXTflex Bisulfite-Seq Kit (BIOO Scientific, Austin, TX, USA) according to the manufacturer’s instructions with minor changes. Briefly, genomic DNA fragments were subjected to end repair, clean-up, and adenylation. Next, barcoded adapters were ligated. The ligation products were purified and size-selected with Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA). Prepared adapter-ligated fragments were subjected to bisulfite conversion with the EZ DNA Methylation-Lightning Kit (Zymo Research, Irvine, CA, USA) according to manufacturer’s protocol, and eluted in 17 µL of M-Elution buffer. Five microliters of converted DNA was amplified in 50 µL volume of PCR reaction (1.25 U of EpiMark Hot Start Taq DNA Polymerase, 1× dedicated buffer, 200 µM dNTPs and 2 µL of NEXTflex Primer Mix) under 1 min of initial denaturation in 95 °C, followed by 18 cycles of 30 s of denaturation in 95 °C 30 s of annealing in 65 °C, and 45 s of elongation in 68 °C, then 5 min of final elongation in 68 °C was applied. Amplicons were purified twice with Agencourt AMPure XP (Beckman Coulter, Brea, CA, USA). The quality of the prepared WGBS libraries was analyzed using Agilent Bioanalyzer and the Agilent High Sensitivity DNA Kit (Agilent Technologies, Santa Clara, CA, USA). Their concentrations were quantified using the Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). For cluster generation, WGBS libraries were pooled with equimolar concentrations, and sequenced using the Illumina HiSeq 4000 system (Illumina, San Diego, CA, USA) in 2× 76 cycles of paired-end (PE) mode with 11 barcoded samples per lane.