GSM5200892: lexA51(Def) ΔumuDC596::ermGT ΔdinB61::ble ΔrnhB782 ::kan with pJM963 replicate 4; Escherichia coli; OTHER
DNA was extracted using MasterPure™ Complete DNA and RNA Purification Kit (Lucigen, Epicentre, MC85200) and its concentration was measured using Qubit® dsDNA BR Assay Kit (Life technologies, molecular probes, Q32853). 1 µg extracted DNA was mixed with 0.3 M KOH and incubated at 55°C for 2 hours. After ethanol precipitation, the pellet was dissolved in 20 µl elution buffer (Qiagen, 1014609). DNA was denatured by 3 min incubation at 85°C. In the phosphorylation step, 5'-OH end generated after KOH hydrolysis at the ribonucleotide site was phosphorylated using a mix of 10 U 3'-phosphatase minus T4 polynucleotide kinase (BioLabs, M0236L), 2.5 µl of 10X reaction buffer (BioLabs, B0201S), and 2.5 µl of 10 mM adenosine 5'-triphosphate (BioLabs, P0756S) in a total volume of 25 µl. After 30 min incubation at 37°C, the reaction was stopped by 20 min incubation at 65°C. The samples were bead-washed using CleanPCR kit (GC biotech, CPCR-0050). In the ligation step, ARC140 adaptor was ligated to the 5'-OP. Denatured DNA was ligated using a mix of 10 U T4 RNA Ligase 1 (ssRNA Ligase) (BioLabs, M0204S), 5 µl of 10X reaction buffer (BioLabs, B0216S), 0.5 µl of 2 mM adenosine 5'-triphosphate (BioLabs, P0756S), 25 µl PEG8000(50%), 5 µl of 10 mM [Co(NH3)6]Cl3, and 0.5 µl of 100 µM oligo ARC140 in a total volume of 50 µl. After overnight incubation at the room temperature, the samples were twice beads-washed and dissolved in 14 µl elution buffer. In second strand synthesis step, denatured DNA was polymerized at the room temperature for 5 min using a mix of 4 U T7 DNA polymerase (unmodified) (BioLabs, M0274L), 2 µl of 10X reaction buffer (BioLabs, B0274A), 2 µl of 2 mM adenosine 5'-triphosphate (BioLabs, P0756S), 0.8 µl of BSA (1 mg/ml) (BioLabs, B9000S), and a heterodimer oligo, ARC76/77 in a total volume of 20 µl. After beads-wash and elution, the libraries were amplified by PCR reactions using KAPA HiFi hotstart readymix (2X) kit (KAPA biosystems, KK2602, 07958935001) with PCR parameters as follows: 95 °C for 45 s, followed by 18 cycles at 95 °C for 15 s, 65 °C for 30 s, and 72 °C for 30 s. The amplified libraries were beads-washed and eluted in 20 µl TE. The library concentration was measured using Qubit® dsDNA HS Assay Kit (Life technologies, molecular probes, Q32854). The library quality was checked by the Agilent 2100 bioanalyzer system (Agilent Technologies). Illumina NextSeq 500 system was used for paired-end sequencing.