CpG Methyltransferase (M.SssI)

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	S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.

Protocol tips
S-adenosylmethionine (SAM) is unstable at (pH 7.5), 37°C and should be replenished in reactions incubated longer than 4 hours.

Publication protocol

Culture conditions and genomic DNA isolation
wild-type strain was grown anaerobically in liquid low osmolarity complex growth (LOC) medium () (final pH 7.0) with maltose (0.5% w/v; Sigma M5895) as the carbon source. Liquid cultures were grown from a 0.5% inoculum and incubated at 75°C in anaerobic culture bottles degassed with five cycles of vacuum and argon. Genomic DNA of was prepared from 50 ml cultures grown to mid-log phase (∼0.1 at OD), harvested by centrifugation at 6000 × at 4°C for 15 min and resuspended in 800 μl of Genomic Lysis buffer (Zymo Research). Cells were lysed by a combination of three freeze-thaw cycles and sonication on ice. The following steps were performed using the Quick-gDNA™ MiniPrep (Zymo Research) according to the manufacturer's instructions. Genomic DNA concentrations were determined using the Qubit 2.0 fluorometer (Invitrogen) and the quality of DNA was assessed by agarose gel electrophoresis.

Preparation of 304 bp model DNA with 4mC modifications
For -methylcytosine (4mC) containing model DNA, 0.5 ng of pUC19 vector DNA (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl -methyl-dCTP (4mdCTP) (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GAACGAAAACTCACGTTAAGGG), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). Cycling parameters: 94°C 2 min, 30 cycles of 94°C 1 min, 37°C 2 min, 55°C 3 min, followed by 55°C 7 min. The PCR product was purified by gel electrophoresis.

Generation of spike-in controls for 4mC-TAB-seq and MethylC-seq of
C/5mC spike-in control (CpG methylated lambda DNA) was prepared by treating unmethylated lambda cl857 DNA (Promega) with M. (NEB) according to the manufacturer's instructions. The CpG methylation was confirmed by standard bisulfite sequencing. To generate the 4mC spike-in control, 0.5 ng of pUC19 vector (NEB) was PCR amplified as follows in a 50 μl reaction: 2.5 U RedTaq polymerase (Sigma), 5 μl 10× reaction buffer, 1 μl 4mdCTP (Trilink)/dATP/dGTP/dTTP cocktail (10 mM each), 1 μl 10 mM forward primer (5′-GCGGTAATACGGTTATCCAC), 1 μl 10 mM reverse primer (5′-TGCTGATAAATCTGGAGCCG). Cycling parameters: 94°C 2 min, 35 cycles of 94°C 1 min, 37°C 2 min, 55°C 5 min, followed by 55°C 7 min. The PCR product was purified by gel electrophoresis.

Condition test using 4mC or 5mC-containing model DNA
The 304 bp DNA with 4mC and CpG methylated lambda DNA were prepared as described above. The recombinant catalytic domain of mouse Tet1 protein (Tet) was expressed and purified as previously described (). Oxidation reactions were performed in a 50 μl solution with 50 mM HEPES buffer (pH 8.0), 100 μM ammonium iron (II) sulfate, 1 mM α-ketoglutarate, 2 mM ascorbic acid, 2.5 mM DTT, 100 mM NaCl, 1.2 mM ATP, 6 ng/μl sonicated mouse embryonic stem cells (mESC) genomic DNA with 0.5% (w/w) 4mC or 5mC-containing model DNA and 4.5 μM Tet. The reactions were incubated at 37°C for 1.5 h. After proteinase K treatment, the oxidized DNA was purified with Micro Bio-Spin 30 Columns (Bio-Rad) and then by QIAquick PCR Purification Kit (QIAGEN). The untreated or Tet-treated mESC genomic DNA with 4mC or 5mC-containing model DNA was applied to MethylCode Bisulfite Conversion Kit (Invitrogen) using thermal cycling program: (i) 98°C 10 min, 64°C 2.5 h or (ii) 98°C 10 min, 53°C 30 min, 8 cycles of 53°C 6 min and 37°C 30 min. After bisulfite conversion, 3 μl of purified converted DNA was PCR amplified using ZymoTaq DNA polymerase (Zymo Research) following the manufacturers’ instructions (for 4mC model, forward primer: 5′-GAATGAAAATTTATGTTAAGGG; reverse primer: 5′-ATTTAAAACTTCATTTTTAATTTAAAA; for 5mC model, forward primer: 5′-TTTGGGTTATGTAAGTTGATTTTATG; reverse primer: 5′-CACCCTACTTACTAAAATTTACACC). The PCR products were TOPO cloned using the TOPO TA cloning kit (Invitrogen), and individual clones were subjected to Sanger sequencing using the M13 Forward primer.

Quantification of 4mC and 5mC in by LC-MS/MS
Five hundred nanograms of genomic DNA was digested by 2 U Nuclease P1 (Wako) in 30 μl solution containing 0.01 M NHAc (pH 5.3) and 2 mM ZnCl at 42°C overnight. After adding 3.5 μl freshly prepared 1 M NHHCO and 1 mU Phosphodiesterase I (Sigma P3134), the reaction was allowed to incubate at 37°C for 2 h followed by addition of 2 U Alkaline Phosphatase (Sigma) and another 2 h incubation at 37°C. The digested sample was filtered and 5 μl was subject to LC–MS/MS. The separation of nucleosides was performed using Agilent 1290 UHPLC system with a C18 reversed-phase column (2.1 × 50 mm, 1.8 μm). The mobile phase A was water with 0.9 ppm (v/v) formic acid (final pH 4.5) and mobile phase B was methanol with 0.9 ppm (v/v) formic acid. Online mass spectrometry detection was performed using an Agilent 6460 triple quadrupole mass spectrometer in positive electrospray ionization mode. Quantification was accomplished in multiple reaction monitoring (MRM) mode by monitoring the transitions of 228.2→112.1 (dC), 242.2→126.1 (4mdC/5mdC). 4mdC and 5mdC had the different retention times. dC, 4mdC and 5mdC were quantified basing on the corresponding calibration curves generated with pure standards.



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