Whole-Genome Bisulfite Sequencing Protocol

DNA was extracted using the DNeasy Plant Mini Kit (Qiagen, Hilden, D) according to the manufacturer’s protocol from segregant plant pools, and quantified using the Qubit dsDNA BR assay kit. Illumina sequencing library preparation and bisulfite conversion was conducted as described in Kawakatsu [55 (link)]. Briefly, DNA was End Repaired using the End-It kit (Epicentre, Madison, WI), A-tailed using dA-Tailing buffer and 3μL Klenow (3’ to 5’ exo minus) (NEB, Ipswich, MA) and Truseq Indexed Adapters (Illumina, San Diego, CA) were ligated overnight. Bead purified DNA was quantified using the Qubit dsDNA BR assay kit, and stored at -20°C. At least 450ng of adapter ligated DNA was taken into bisulfite conversion which was performed according to the protocol provided with the MethylCode Bisulfite Conversion kit (Thermo Fisher, Waltham, MA). Cleaned, converted single stranded DNA was amplified by PCR using the KAPA U+ 2x Readymix (Roche Holding AG, Basel, CH): 2 min at 95°C, 30 sec at 98°C [15 sec at 98°C, 30 sec at 60°C, and 4 min at 72°C] x 9, 10 min at 72°C, hold at 4°C. After amplification, the DNA was bead purified, the concentration was assessed using the Qubit dsDNA BR assay kit, and the samples were stored at -20°C. WGBS library was sequenced as part of a large multiplexed pool paired-end 150 bp on an Illumina HiSeq 4000.