Genome-wide Methylome Mapping via Azido-Glucose Chemical Tagging

Genomic DNA was sheared into fragments with an average size of 200 bp using an M220 Focused-Ultrasonicator (Covaris) according to the manufacturer’s instructions. The fragmented DNA was measured using an Agilent 4200 TapeStation System (Agilent). Four micrograms of fragmented DNA from HEK293T cells or 50 ng fragmented DNA from tumor tissues were treated with T4 Phage β-glucosyltransferase (New England Biolabs; Catalog #M0357S) and 60 μM UDP-6-azide-glucose (Jena Bioscience) in 1X NEBuffer™ 4 (New England Biolabs; Catalog #B7004S) at 37 °C for 1 h. Then, 2 μL DBCO-PEG4-Biotin Conjugate (Jena Bioscience, 20 mM stock in DMSO) was added to the reaction mixture and incubated at 37 °C for 2 h. The modified DNA was then purified using AMPure XP beads. Next, modified Illumina-compatible adapters (Additional file 6: Table S1) (BIONEER) were ligated to the DNA fragments using a NEBNext^® Ultra™ II DNA Library Prep Kit for Illumina^® (New England Biolabs; Catalog #E7645S) according to the manufacturer’s protocol. The adapter-ligated DNA was then treated with T4 Phage β-glucosyltransferase (New England Biolabs; Catalog #M0357S) and UDP-Glucose in 1X NEBuffer supplied with the enzyme in a total reaction volume of 16 μL at 37 °C for 1 h. Next, 4 μL formamide was added, and the mixture was incubated at 80 °C for 10 min and then immediately put on ice. The denatured DNA was then subjected to enzymatic deamination using a NEBNext^® Enzymatic Methyl-seq Kit (New England Biolabs; Catalog #E7120S). The converted DNA was then incubated with 5 μl Dynabeads™ MyOne™ Streptavidin C1 (Invitrogen) in 1X binding and washing buffer (5 mM Tris–HCl (pH 7.5)) 500 μM EDTA, 1 M NaCl) at room temperature for 15 min. The beads were then washed with 1X binding and washing buffer five times. HEK293T samples underwent another round of enzymatic deamination for complete conversion of cytosines and 5-methyl cytosines. Enriched DNA was amplified with 12 cycles of PCR using NEBNext Q5U Master Mix supplied in the NEBNext^® Enzymatic Methyl-seq Kit. Libraries were sequenced on an Illumina Novaseq6000 platform to generate paired-end data.