MA Line 49 replicate 1
Approximately two micrograms of genomic DNA was sonicated to ~100 bp using the Covaris S2 System using the following parameters: cycle number = 6, duty cycle = 20%, intensity = 5, cycles/burst = 200 and time = 60 seconds. Sonicated DNA was purified using Qiagen DNeasy minielute columns (Qiagen). Each sequencing library was constructed using the NEBNext DNA Sample Prep Reagent Set 1 (New England Biolabs, Ipswich, MA) according to the manufacturer’s instructions with the following slight modifications. Methylated adapters were used in place of the standard genomic DNA adapters from Illumina (Illumina, San Diego, CA). Ligation products were purified with AMPure XP beads (Beckman, Brea, CA). DNA (450 ng) was bisulfite treated using the MethylCode Kit (Invitrogen, Carlsbad, CA) following the manufacturer’s guidelines and then PCR amplified using Pfu Cx Turbo (Agilent, Santa Clara, CA) instead of using the Phusion Taq included in the NEBNext kit using the following PCR conditions (2 minutes at 95C, 4 cycles of 15 seconds at 98C, 30 seconds at 60C, 4 minutes at 72C and 10 minutes at 72C).