Oxford Nanopore sequencing of Saccharomyces cerevisiae: dnCas3-APOBEC1_target 2
Per strain 30 replicates were sequenced. For each replicate two overlapping ~6 kb amplicons were generated, covering a region of ~12 kb downstream of the respective target site (Chr.V: 21741-33687 for target site 1 and Chr.V: 22595-34278 for target site 2). PCR amplification was done using Q5 Hot-Start High Fidelity DNA Polymerase (NEB). The presence and quality of each amplicon was examined via absorbance measurements on NanoDrop 8000 and visually, via gel electrophoresis. The two ~6 kb amplicons per sample were pooled and sent for Nanopore sequencing at the Neuromics Support Facility, VIB-UAntwerp Center for Molecular Neurology. PCR products were purified using AMPure XP reagent (Beckman Coulter), ratio 0.8x. Purification was performed on an automated platform (Beckman Coulter Biomek FxP). Removal of smaller molecules (e.g. primer dimers) was checked using the Agilent Fragment Analyzer 5300 and the DNF-492 Standard Sensitivity Large Fragment analysis kit. Next, each sample/amplicon was barcoded using the PCR Barcoding Expansion 1-96 kit (EXP-PBC096, Oxford Nanopore Technologies (ONT)) and the LongAmp Taq 2X Master Mix (M0287L, New England Biolabs). Barcoded samples/amplicons were purified using AMPure XP reagent (Beckman Coulter), ratio 0.8x. Purification was performed on an automated platform (Beckman Coulter Biomek FxP). Amplicons were analyzed using the Agilent Fragment Analyzer 5300 and the DNF-492 Standard Sensitivity Large Fragment analysis kit. Amplicon concentrations of each sample was quantified via Qubit 1x dsDNA High Sensitivity (Thermo Fisher Scientific). Samples were pooled in equal amounts and the resulting pool was purified using AMPure XP reagent and eluted in nuclease-free water. The purified pool was again analyzed using the Agilent Fragment Analyzer 5300 and the DNF-492 Standard Sensitivity Large Fragment analysis kit. The pooled samples were subjected to ONT library preparation using the SQK-LSK110 Ligation Sequencing kit (ONT) (input 100 fmol/pool). End repair was carried out with NEBNext Ultra II End Repair/dA-Tailing Module (E7546L, New England Biolabs). The pool was purified using AMPure XP reagent (Beckman Coulter), ratio 1x and eluted in nuclease-free water. The concentration was quantified via Qubit 1x dsDNA High Sensitivity (Thermo Fisher Scientific). Next, adapters were added to end repaired DNA using NEBNext Quick Ligation Module (E6056L, New England Biolabs). NEBNext Quick Ligation Reaction Buffer was replaced by ONT proprietary Ligation Buffer. The pool was purified using AMPure XP reagent (Beckman Coulter), ratio 1x and eluted in Buffer EB (ONT). The concentration was quantified via Qubit 1x dsDNA High Sensitivity (Thermo Fisher Scientific). Finally, 10 fmol of the pool were loaded on a R9.4.1 Flongle Flow Cell (FLO-FLG001, Oxford Nanopore Technologies) with >50 available pores after Flow Cell QC. For 61 replicates the sequencing was done twice. The resulting bam files contain the addition _v2 in their file name.