Profiling 5-Hydroxymethylcytosine in Tumor DNA

50 ng fragmented DNA from tumor tissues were treated with T4 Phage β-glucosyltransferase (New England Biolabs; Catalog #M0357S) and UDP-6-azide-Glucose (Jena Bioscience) in 1X NEBuffer supplied with the enzyme (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, TruSeq adapters 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 DNA fragments with 5hmC were enriched as described above. 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.