title: targeted SMRT-TAPS of E14 mESCs: chr13:101123190-101127596
design: 1 ng of model DNA or 50 ng of genomic DNA sample was incubated in a 20 L reaction containing 50 mM HEPES buffer (pH 7.0), 100 M ammonium iron(II) sulfate, 1 mM -ketoglutarate, 2 mM ascorbic acid, 1 mM dithiothreitol, 100 mM NaCl, 1.2 mM ATP and 4 M hTet2 for 80 min at 30C. Then 0.8 U of Proteinase K (New England Biolabs) were added to the reaction and incubated at 50C for 1 h. After cooling down to room temperature, 6 L of 3 M sodium acetate solution (pH = 4.3) and 3 L of 10 M pyridine borane (Sigma) were added to the reaction mixture directly and incubated at 37C 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).