Encapsulated DNA from E. coli containing plasmid pACBSR-OH and pRW50
Isolated DNA was sonicated for 3 min in an ultrasonic water bath at room temperature. Sequencing libraries were prepared according to the in-house protocol for DNA library preparation. DNA end-repair was performed in 40 l using T4 polynucleotide kinase and T4 DNA polymerase at 25C for 25 min, followed by incubation at 12 C for 10 min. The reaction was purified with 1.8 volumes of MagSI-NGSPREP Plus magnetic beads and eluted with 20 l of dH2O. P1 and A adaptors with corresponding barcodes were ligated to the ends of the DNA fragments using T4 DNA ligase and Bst 2.0 WarmStart DNA polymerase. 30 l reactions were incubated for 30 min at 22 C followed by incubation for 20 min at 50 C. Reactions were purified with 1.8 volumes of MagSI-NGSPREP Plus magnetic beads and eluted with 20 l of dH2O. The library was amplified (50 l) with 6 cycles using KAPA HiFi HotStart DNA Polymerase and P1amp and T_PCR_A primers (0.5 M) using the following amplification protocol: initial denaturation 95 C 3 min followed by 6 cycles 98 C 20 sec, 60 C 30 sec and 72 C 30 sec. The PCR reaction was purified with 1.8 volumes of MagSI-NGSPREP Plus magnetic beads and eluted with 20 l of dH2O. Libraries were checked for size and concentrations according to the Agilent High Sensitivity Kit protocol. Equimolar amounts of libraries were pooled together and amplified according to the protocol of the Ion PI Hi-Q OT2 200 kit. Ion Proton System and samples were prepared according to the protocol of the Ion PI Hi-Q Sequencing 200 kit and sequenced on an Ion PI Chip v3. Basecalling was performed from the device pipeline and adapters were removed from the raw sequencing data.