Bs seq

10 ng of gDNA ligated to 5mC- or 5pyC-containing adapters was used as input. A methylated copy-strand was created. First, 1 μM of copy primer was annealed (Supplementary Table 1, v1), in a total volume of 10 μL in CutSmart Buffer and 1 mM final concentration individually of dATP/dGTP/dTTP (Promega) and dmCTP (NEB). 5 units of Klenow (exo-) polymerase (NEB) was then added and incubated for 30 min at 37°C. After purification (Zymo Oligo Clean & Concentrator), libraries were mixed with 1 μM untagged M.MpeI-N374K and 160 μM CxSAM in carboxymethylation buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM EDTA) and incubated for 1 hour at 37°C followed by denaturation for 5 min at 95°C. 1 μL of Proteinase K was added (NEB) and samples were incubated at 37°C for 15 min. The samples were purified using SPRI beads (1.2x) and eluted in 1 mM Tris-Cl, pH 8.0. DNA was then subjected to BS-Seq (Diagenode, see below) or to snap-cooling and A3A deamination in a final volume of 50 μL as previously described ^{25 (link)}. Purified DNA was then amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA) before purification over SPRI beads (0.8X) to yield final libraries. The non-optimized workflow was used in Figure 2.

Dam^–/Dcm^– lambda phage gDNA was obtained (Thermo Fisher) and quantified by Qubit (Thermo Fisher). All gDNA was sheared to ~350 bp using a Covaris sonicator before SPRI purification (Beckman, 1.2x). DNA was then end-repaired with NEBNext Ultra End Prep Kit and ligated to 5mC-containing xGEN Y-shaped adapters (IDT). 10 ng 5mC-adapter ligated gDNA was reacted with untagged 0.5 μM M.MpeI-N374K and 160 μM CxSAM in M.MpeI reaction buffer (50 mM NaCl, 10 mM Tris-HCl pH 7.9, 10 mM EDTA) and incubated for 1 hour at 37°C followed by denaturation for 5 min at 95°C. 1 μL of Proteinase K was added (NEB) and incubated at 37°C for 15 min. The samples were SPRI purified (1.2x) and subjected to standard BS-Seq (Diagenode, see below). The library was amplified using indexing primers (IDT) and HiFi HotStart Uracil+ Ready Mix (KAPA) before purification over SPRI beads (0.8X) to yield final libraries.