GSM6595703: E.coli rep1 H; Escherichia coli K-12; OTHER
Genomic DNA from Escherichia coli strain K-12 was purchased from ATCC (10798D-5). All the DNA samples were harshly treated with RNase A (NEB, T3018L) again prior to libraries construction to get rid of any RNA contaminants. For E.coli DNA and mtDNAs, 400 ng~1 μg RNA-free DNA was diluted by TE buffer (Invitrogen, AM9849) with 0.1 M NaOH and sonicated using Bioruptor Pico sonication device to 100~300 nt length. The fragmented DNA was mixed with 15% (mass percentage) spike-in probes (Supplementary Table 1) and then spited into three in the proportion of 3:4:1, each for mutation group, demethylation control and high-fidelity control. The three groups of DNAs was denatured and subjected to splint ligation following a previous protocol (Supplementary Table 1) [10.1093/nar/gkw1110]. The ligation products of mutation group and demethylation control underwent primer extension at 60℃ for 3 hours in the 50 μL reaction containing 6 μM biosample-primer-extension primer (Supplementary Table 1), 1200 units of Bst 2.0 DNA polymerase (NEB, M0537M), 1× EpiMark Hot Start Taq Reaction Buffer (NEB, M0490S), 8mM dATP (NEB, N0446S), 2mM dCTP (NEB, N0446S), 2mM dGTP (NEB, N0446S), 0.02 mM 2-thio-dTTP (Trilink, N-2035), 6 mM magnesium sulfate (NEB) and 8% PEG4000 (Rigaku, # 25322-68-3). The resulting libraries were purified by gel electrophoresis and recovery, and finally sequenced at 60~100M depth with single-ended 100 bp mode.