Udp 6 azide glucose

Neuroblastoma cells were grown for 72 h at atmospheric or hypoxic conditions, and the extracted gDNA was sonicated on M220 Focused-ultrasonicator (Covaris, Woburn, MA, United States) in 10 mM Tris-HCl (pH 8.5) buffer to yield fragments with a peak size of ∼200 bp. 5hmC glycosylation was carried out in a 100-μl reaction mixture with 800–1,000 ng fragmented gDNA supplemented with 50 μM UDP-6-azide-glucose (Jena Bioscience) and 10 U T4 β-glucosyltransferase (TS) for 2 h at 37°C followed by enzyme inactivation at 65°C for 20 min and column purification [GeneJet PCR Purification kit (TS)]. Azide-tagged DNA was processed as described previously (Gibas et al., 2020 (link)). DNA libraries were amplified for 12 cycles, size selected for ∼300 bp fragments (MagJET NGS Cleanup and Size Selection kit, TS), and subjected to Ion Proton (TS) sequencing.

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.