NOMe-seq/dSMF experiments were carried out as previously described [41 (link)], with some modifications. Cells were pelleted at 10,000 g, then crosslinked as described for ATAC-seq at 1% formaldehyde concentration.
Fixed cells were resuspended in 100 μL M.CviPI Reaction Buffer (50 mM Tris–HCl pH 8.5, 50 mM NaCl, 10 mM DTT), then treated with M.CviPI by adding 200 U of M.CviPI (NEB), SAM at 0.6 mM and sucrose at 300 mM, and incubating at 30 °C for 20 min. After this incubation, 128 pmol SAM and another 100 U of enzyme were added, and a further incubation at 30 °C for 20 min was carried out. For dSMF experiments, M.SssI treatment followed immediately, by adding 60 U of M.SssI (NEB), 128 pmol SAM, and MgCl2 at 10 mM and incubation at 30 °C for 20 min. The reaction was stopped by adding an equal volume of Stop Buffer (20 mM Tris–HCl pH 8.5, 600 mM NaCl, 1% SDS, 10 mM EDTA).
Crosslinks were reversed overnight at 65 °C, and DNA was isolated using the MinElute PCR Purification Kit (Qiagen, Cat # 28,006).
Enzymatically labeled DNA was then sheared on a Covaris E220, and converted into sequencing libraries following the EM-seq protocol, using the NEBNext Enzymatic Methyl-seq Kit (NEB, Cat # E7120L).
SMF/NOMe-seq libraries were sequenced as 2 × 150mers on a NovaSeq S4 through Novogene to a depth of ∼100 × for 200-bp fragments.
Fixed cells were resuspended in 100 μL M.CviPI Reaction Buffer (50 mM Tris–HCl pH 8.5, 50 mM NaCl, 10 mM DTT), then treated with M.CviPI by adding 200 U of M.CviPI (NEB), SAM at 0.6 mM and sucrose at 300 mM, and incubating at 30 °C for 20 min. After this incubation, 128 pmol SAM and another 100 U of enzyme were added, and a further incubation at 30 °C for 20 min was carried out. For dSMF experiments, M.SssI treatment followed immediately, by adding 60 U of M.SssI (NEB), 128 pmol SAM, and MgCl2 at 10 mM and incubation at 30 °C for 20 min. The reaction was stopped by adding an equal volume of Stop Buffer (20 mM Tris–HCl pH 8.5, 600 mM NaCl, 1% SDS, 10 mM EDTA).
Crosslinks were reversed overnight at 65 °C, and DNA was isolated using the MinElute PCR Purification Kit (Qiagen, Cat # 28,006).
Enzymatically labeled DNA was then sheared on a Covaris E220, and converted into sequencing libraries following the EM-seq protocol, using the NEBNext Enzymatic Methyl-seq Kit (NEB, Cat # E7120L).
SMF/NOMe-seq libraries were sequenced as 2 × 150mers on a NovaSeq S4 through Novogene to a depth of ∼100 × for 200-bp fragments.