title: GSM8599782: dcm-/dam- lambda phage DNA, M.SssI, bisulfite treated; Escherichia phage Lambda; Bisulfite-Seq
library_construction_protocol: The Whole-genome Bisulfite Sequencing (WGBS) libraries were prepared as follows. For A-tailing, 200 ng sheared DNA (~ 300 bp) in 25 µl ddH2O (MilliQ), 3.5 µl NEBNext Ultra II End Prep Reaction Buffer (NEB, cat. no. E7546L) and 1.5 µl NEBNext Ultra II End Prep Enzyme Mix (NEB, cat. no. E7546L) were mixed by pipetting and incubated at 20°C for 30 min. The reaction was inactivated by incubating at 65°C for 30 min. Subsequently, the DNA was purified with 1.8x MagMAX™ Pure Bind beads (ThermoFisher, cat. no. A58522) and the DNA was eluted at 37°C in 16 µl prewarmed (at 50°C) ddH2O (MilliQ) for 5 min. For adapter ligation, 15 µl A-tailed DNA, 2 µl 10x T4 DNA Ligase Buffer (NEB, cat. no. B0202S), 1.5 µl T4 DNA Ligase 2,000 units/µl (NEB, cat. no. M0202M), 1 µl 10 mM ATP (NEB, cat. no. P0756S) and 0.5 µl 50 mM TruSeq Adapter (Microsynth) were mixed by pipetting and incubated at 16°C overnight. The finished ligation reaction was directly subjected to bisulfite treatment according to the manufacturer's protocol using the EZ DNA Methylation-Gold™ Kit (Zymo Research, cat. no. D5005). The DNA was eluted on the bench in 15 µl prewarmed (55°C) M-Elution Buffer (Zymo Research) for 5 min.