Pyridine borane

Ligated DNA was added to a 50-μL reaction containing 50 mM HEPES buffer (pH 8), 25 mM MgCl₂, 200 μM UDP-Glc (NEB), and 10 U of βGT (Thermo Fisher) for 1 h at 37 °C. 5hmC-blocked DNA was purified with Ampure XP and then incubated in 50 µL oxidation reaction containing 50 mM HEPES buffer (pH 8.0), 100 µM ammonium iron (II) sulfate, 1 mM α-ketoglutarate, 2 mM ascorbic acid, 1 mM dithiothreitol, 100 mM NaCl, 1.2 mM ATP, and 4 μM mTet1CD for 80 min at 37 °C. Then 0.8 U of Proteinase K (NEB) was added to the reaction and incubated for 1 h at 50 °C. Oxidized DNA was purified with Ampure XP beads and then input into another round of TET oxidation in order to achieve complete oxidation. The double-oxidized DNA was added to a 50-μL reaction containing 600 mM NaAc (pH = 4.3) and 1 M pyridine borane (Alfa Aesar). The reaction was incubated at 37 °C and 850 r.p.m. in a ThermoMixer (Eppendorf) for 16 h and purified by Zymo-IC column (Zymo Research) with Oligo Binding Buffer (Zymo Research).

One nanogram of model DNA or 50 ng of genomic DNA sample was incubated in a 20 μL reaction containing 50 mM HEPES buffer (pH 7.0), 100 μM ammonium iron(II) sulfate, 1 mM α-ketoglutarate, 2 mM ascorbic acid, 1 mM dithiothreitol, 100 mM NaCl, 1.2 mM ATP, and 4 μM hTet2 for 80 min at 30 °C. Then 0.8 U of Proteinase K (NEB) were added to the reaction and incubated at 50 °C for 1 h. After cooling down to room temperature, 6 μL of 3 M sodium acetate solution (pH = 4.3) and 3 μL of 10 M pyridine borane (Alfa Aesar) were added to the reaction mixture directly and incubated at 37 °C and 850 rpm in a ThermoMixer (Eppendorf) for 16 h. The reaction was purified with Zymo-IC column (Zymo Research) and Oligo Binding buffer (Zymo Research). The converted DNA was then amplified with LongAmp Hot Start Taq 2X Master Mix (NEB). The detailed protocol is described in Additional file 1: Supplementary method 2. Primer sequences are listed in Additional file 1: Table S2.