Instrument: Illumina NovaSeq 6000
Strategy: Bisulfite-Seq
Source: GENOMIC
Selection: RANDOM
Layout: PAIRED
Construction protocol: D. radiodurans DNA was isolated using Ezup Column Bacteria Genomic DNA Purification kit (Sangon, Shanghai, China) according to the manufacturer's instruction. D. radiodurans DNA (100 ng in 400 μL of water) was fragmented to an average size of 300–400 bp using an ultrasonic homogenizer. The sheared DNA was lyophilized to dryness and then reconstituted in water. The resulting DNA was end-repaired and adenylated at 30°C for 20 min and immediately at 72°C for 20 min using Hieff NGS® Ultima Endprep Mix (Yeasen Biotechnology Co., Ltd., Shanghai). Adaptor-OH and Adaptor-P (Table S4 in Supporting Information) were mixed at a final concentration of 20 μM each in 1× annealing buffer and then incubated in 95°C for 5 min followed by cooling gradually to 25°C. Adaptor ligation was performed at 20°C for 15 min using Hieff NGS® Ultima DNA Ligation Module (Yeasen) followed by DNA purification using KAPA Pure beads (Roche). The adaptor ligation was confirmed by 20% native PAGE. The resulting DNA (16 μL) was added with 2 μL of DMSO and then denatured at 95°C for 10 min and immediately chilled at 0oC. The deamination reaction by A3A protein was carried out at 37°C for 2 h in a final concentration of 20 mM MES (pH 6.5), 0.1% Triton X-100 and 6 μM A3A protein. The resulting A3A-deaminated DNA was amplified by PCR with 10 cycles using pre-P5 primer, pre-P7 primer, and Q5U® Hot Start High-Fidelity DNA polymerase (New England Biolabs). After purification by KAPA Pure beads, the DNA products were amplified by PCR with 5 cycles using P5-universal primer, P7-index primer, and Q5® Hot Start High-Fidelity 2× Master Mix (New England Biolabs). The final PCR products were purified with KAPA Pure beads and 1.5% agarose gel electrophoresis and examined by Agilent 2100 before sequencing on the Illumina NovaSeq 6000 platform (Novogene Co., Ltd., China).