ONT-seq of Lispe tenatculata
Two libraries were prepared simultaneously. The input DNA was prepared by transferring 1 g of gDNA into a 0.2 ml PCR tube and adjusted with nuclease-free water to 47 l. The initial binding incubation time at 65C, all incubation times during a bead-based AMPure XP clean-up after DNA repair, end-prep, and adapter ligation steps, as well as incubation with a NEBNext Quick T4 DNA Ligase (New England BioLabs Inc., Ipswich, MA, USA) at room teperature were doubled. For the beads washing step that follows the adapter ligation, the Long Fragment Buffer (LFB) was used. The incubation in Elution Buffer (EB) was performed at 37C. After the completion of the library preparation protocol, one library was loaded onto a SpotON Flow Cell Rev D (R.9.4.1; FLO-MIN106D) and the second library was stored in 4C according to the manufacturer's recommendations. Two sequencing runs on one flow cell were performed using a MinION Mk1C device (MIN-101C) (Oxford Nanopore Technologies). After the first run, the flow cell was washed with the Flow Cell Wash Kit (EXP-WSK002) following the manufacturers protocol and immediately the second library was loaded for sequencing.