Nextflex bisulfite library preparation kit

RRBS libraries were performed using the NEXTflex Bisulfite Library Preparation Kit (Bioo Scientific, Austin, TX, USA) according to manufacturer's instructions. In brief, 500 ng of high-molecular-weight genomic DNA was used for MspI digestion (New England Biolabs, Ipswich, MA, USA) followed by DNA purification using Agencourt AMPure XP magnetic beads (Beckman Coulter, Beverly, MA, USA). Purified fragmented DNA samples with 5 0 -CG-3 0 overhangs were subject into end repair, adenylation, and adaptor ligation reactions. Bisulfite conversion was performed using the EZ DNA Lightning Kit (Zymo Research, Irvine, CA, USA), according to the manufacturer's instructions, and it was followed by PCR amplification. The PCR products were purified using Agencourt AMPure XP magnetic beads (Beckman Coulter). Prior to sequencing, RRBS libraries were quantified using Qubit instrument (Thermo-Fisher Scientific, San Jose, CA, USA) and qualified using Agilent 2100 Bioanalyzer High Sensitivity chips (Agilent Technologies, Santa Clara, CA, USA). Paired-end sequencing (2 3 100 bp) was performed on the HiSeq 1500 platform (Illumina, Scientific, San Diego, CA, USA).