Design Description | Total genomic DNA (~5 ?g) was fragmented to 1001000 bp using Bioruptor (Diagenode, Denville, New Jersey). End repair (NEBNextŽ End Repair Module) was performed on the DNA fragment followed by adding an 'A' base to the 3' end (NEBNextŽ dA-Tailing Module), and the resulting DNA fragment was ligated to the methylated DNA adapter (NEXTflex DNA Barcodes, Bioo Scientific, Austin, Texas). The adapter-ligated DNA of 200400 bp was purified using AMPure beads (Beckman Coulter, Brea, California), followed by sodium bisulfite conversion using MethylCode Bisulfite Conversion Kit (Life Technologies, Foster City, California). The bisulfite-converted DNA was amplified by 12 cycles of PCR using LongAmpŽ Taq DNA Polymerase (NEB, Ipswich, Massachusetts) and purified using AMPure beads (Beckman Coulter, Brea, California). The Paired-End sequencing of the MethylC-seq libraries was performed for 126 cycles. |