title: GSM6288283: L1710-Haloferax_volcanii_exponential_dSMF; Haloferax volcanii DS2; OTHER
library_construction_protocol: Cells were fixed by adding 37% formaldehyde (Sigma) at a final concentration of 1% and incubating for 15 minutes at room temperature. Formaldehyde was then quenched using 2.5 M Glycine at a final concentration of 0.25 M. Cells were subsequently centrifuged at 10,000 g for 2 minutes, washed once in 1x PBS, and centrifuged again at 10,000 g for 2 minutes. Fixed cells were resuspended in 100 uL 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 30C for 20 minutes. After this incubation, 128 pmol SAM and another 100 U of enzyme were added, and a further incubation at 30C 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, MgCl2 at 10 mM and incubation at 30C 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 65C, and DNA was isolated using the MinElute PCR Purification Kit (Qiagen, Cat \# 28006). 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).