Ecori methyltransferase

1 M Tris-HCl (pH8, molecular biology grade ultrapure, ThermoScientific, cat#J22638-K2), 2-deoxyadenosine (A) (>99%, FisherScientific, cat#AAJ6388606), Benzonase (Millipore Sigma, cat#E1014), CpG methyltransferase M.SssI(NEB, cat#M0226), dNTP set (Neta Scientific, cat#GHC-28-4065-51), HMW DNA Extraction kit (Promega, Cat#A2920), M. SssI (NEB, cat#M0226S), N6-methyl-2-dATP (TriLink, cat#N-2025), N6-methyl-2-deoxyadenine (m6A) (>99%, FisherScientific, cat#AAJ64961MD), Nanosep (MWCO 3 kDa, Pall, cat#OD003C33), phosphodiesterase I (Worthington, cat#LS003926), Quick CIP (NEB, cat#M0525S), Ultrapure distilled water (Invitrogen, cat#10977015). Taq methyltransferase (NEB, cat#M0219S), Dam methyltransferase (NEB, cat#M0222S), EcoRI methyltransferase (NEB, M0211S), ProNex^® Size-Selective Chemistry (Promega, cat#NG2001), PacBio Elution buffer (PacBio #101-633-500), ZORBAX Eclipse Plus C18 (Agilent, cat#959757-902). All reagents used for mass spectrometer analysis are molecular grade level or above. g-TUBEs, SMRTbell^® prep kit 3.0, REPLI-g Mini kit (Cat# 150023).

DNA methylation was performed using a 25-μl mixture containing 0.5 or 1.5 pmol DNA, EcoRI methyltransferase reaction buffer (New England BioLabs; 50 mM Tris-HCl, 50 mM NaCl, and 10 mM ethylenediaminetetraacetic acid [pH 8.0]), 80 μM S-adenosylmethionine (New England Biolabs), and 40 units of EcoRI methyltransferase (New England BioLabs). After incubation for 2.5 h at 37°C, the reaction was terminated by incubation for 20 min at 65°C. Methylated DNA was purified using the high pure PCR product purification kit and dissolved in 100 μl H₂O.