NextSeq 500 sequencing; Sequence preference of DNMT3A/B dictates genome-wide DNA methylation signature
Sequence preference of DNMT3A/B dictates genome-wide DNA methylation signature
DNMT3A-Abcam-120min_s
Full-length recombinant human DNMT3A (ab170408) and DNMT3B (ab170410) were purchased from Abcam. 100 ng unmethylated E. coli genomic DNA (D5016, Zymo research)  was incubated at 37 °C with 500 ng of DNMT3A or DNMT3B and 160 µM S-adenosylmethionine (SAM, Cat. No. B9003S, NEB) in reaction buffer (50 mM Tris-HCI, 1 mM EDTA, 1 mM dithiothreitol, 5% glycerol, 100 µg/ml BSA) for 30, 120 min. DNA was purified using DNA Clean & Concentrator Kit (D4030, Zymo research) and processed for high throughput bisulfite sequencing. Bisulfite library was prepared using Pico Methyl-Seq Library Prep Kit (D5456, Zymo research) per manufacture's protocol. Briefly, methylated DNA and unmethylated E. coli DNA were treated with bisulfite conversion reagent at 98 °C for 8 min, then 54 °C for 60 min.