Whole genome bisulfite sequencing (WGBS)

WGBS single indexed libraries were generated using the NEBNext Ultra DNA library Prep kit for Illumina (New England BioLabs, Ipswich, MA, USA) on the Agilent Bravo automated liquid handling platform with a custom high-throughput protocol. Forty to 300 ng gDNA was quantified by Qubit dsDNA BR assay (Invitrogen, Carlsbad, CA, USA), and 1% unmethylated lambda DNA (cat#: D1521, Promega, Madison, WI, USA) was spiked in to measure bisulfite conversion efficiency. DNA was fragmented to an average insert size of 400–500 bp using the Covaris LE220 Focused-ultrasonicator in a 55-μl volume. The fragmented gDNA was converted to end-repaired, adenylated DNA using the NEBNext Ultra End Repair/dA-Tailing Module (cat#: 7442 L, New England BioLabs, Ipswich, MA, USA). Methylated adaptors (NEBNext Multiplex Oligos for Illumina; cat#: E7535L New England BioLabs, Ipswich, MA, USA) were ligated to the product from the preceding step using the NEBNext Ultra Ligation Module (cat#: 7445 L, New England BioLabs, Ipswich, MA, USA). Size selection was performed using AMPure XP beads and insert sizes of ~ 400 bp were isolated (0.37x and 0.20x ratios). Samples were bisulfite converted after size selection using the EZ-96 DNA Methylation-Gold Kit (cat#: D5008, Zymo, Irvine, CA, USA) following the manufacturer’s instructions. Amplification was performed following bisulfite conversion using primers from the NEBNext Multiplex Oligos for Illumina module (cat#: E7535L, New England BioLabs, Ipswich, MA, USA) and the Kapa HiFi Uracil+ PCR system (cat#: KK2801, Kapa Biosystems, Boston, MA, USA) with the following cycling parameters: 98 °C 45 s/8 cycles: 98 °C 15 s, 65 °C 30 s, 72 °C 30 s/72 °C 1 min. The PCR enriched product was cleaned up using 0.9x AMPure XP beads (cat#: A63881, Beckman Coulter, Brea, CA, USA). Final libraries were run on 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) using the High-Sensitivity DNA assay; samples were also run on Bioanalyzer after shearing and size selection for quality control purposes. Libraries were quantified by qPCR using the Library Quantification Kit for Illumina sequencing platforms (cat#: KK4824, KAPA Biosystems, Boston, MA, USA), using 7900HT Real-Time PCR System (Applied Biosystems). Libraries were sequenced with the Illumina HiSeq4000 using 151-bp paired-end run with a 27% PhiX spike-in.