title: GSM7669525: E.coli WGA rep1 H; Escherichia coli; OTHER
library_name: GSM7669525
library_construction_protocol: 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, 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. The 100-mer biotin-tagged NN-6mA-NN model DNA oligo and primer-extension primer was synthesized by IDT (Integrated DNA Technologies). At first, the primer-extension primer was denatured at 70 for 2 min and immediately chilled on ice. In a total volume of 50 L, 50 ng model DNA and 2.5 L 2 M oligo-primer-extension primer were mixed with different DNA polymerases, such as OneTaq DNA Polymerase, Vent DNA Polymerase, phi29 DNA Polymerase, Pwo SuperYield DNA Polymerase, EpiMark Hot Start Taq DNA Polymerase, Bst DNA Polymerase-full length, Bst 2.0 DNA Polymerase, Bst 2.0 WarmStart DNA Polymerase, Bst 3.0 DNA Polymerase, Recombinant HIV reverse transcriptase), and the supplied buffers, with the supplement of different concentration of magnesium sulfate, manganese chloride, polyethylene glycol (PEG8000, PEG4000, PEG1000, PEG600, PEG600, PEG200), dATP/dCTP/dGTP and modified dTTP/UTP (dUTP, UTP, 2-Thio-dTTP, 4-Thio-dTTP, 5-Aminoallyl -dUTP, Fluorescein-12-dUTP). Primer extension tests were further carried out under different temperatures. The newly synthesized strands by extension were subsequentially released from the biotinylated template strands immobilized on Streptavidin C1 beads (Thermo Fisher, 65001) by incubation in 150 mM NaOH. The released strands were neutralized, cleaned up by Oligo Clean & Concentrator Kit (Zymo Research, D4061), and incubated with Streptavidin C1 beads again for the complete removal of template strands. The supernatant was cleaned up with Oligo Clean & Concentrator Kit and amplified for 10 cycles by NEBNext Ultra II Q5 Master Mix (NEB, M0544S) and NEBNext Multiplex Oligos for Illumina (NEB, E7335S). The resulting libraries were purified by gel electrophoresis and recovery (NEB, T1020S). All libraries were sequenced at 2M reads depth in single-ended 100 bp mode at NovaSeq 6000 sequencer (Illumina). The primer extension condition was finalized as: 1500 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). All reactions were incubated at 60 for 1 h.