Udp 6 n3 glucose

Five μg of genomic DNA was sonicated to 300–400 base pair (bp) using a Covaris focused ultrasonicator. 5hmC capture was performed according to the method described in Song et al. (2011) (link). First, a glucosyltransfer reaction was performed using 2.5μl of T4 phage β-glucosyltransferase enzyme (10,000U/ml; New England BioLabs #M0357L) and 100μM UDP-6-N3-glucose (Jena Biosciences #CLK-076) and incubated at 37°C for 2 hours. After purification with AMPure XP beads (Beckman Coulter A63881), the glucosylated 5hmC-containing DNA fragments were biotinylated with 150μM disulfide biotin linker (Click Chemistry Tools A112–5) at 37°C for 2 hours. After purification with AMPure XP beads, the biotinylated 5hmc-containing DNA fragments were pulled down using Dynabeads MyOne Streptavidin C1 beads (ThermoFisher Scientific #65002) and were washed three times with Binding/Washing buffer (20mM Tris pH 7.5, 1mM EDTA, 2M NaCl, 0.01% Tween-20). The 5hmC-containing DNA fragments were then removed from the beads with fresh 100mM dithiothreitol for 2 hours with rotation at room temperature. After final purification with AMPureXP beads, the 5hmC-enriched DNA fragments were eluted in nuclease-free water and quantified by Qubit.