Cas9 targeted Nanopore sequencing for genomic region covering TP53
extracted HMW gDNA was phosphatase treated to remove pre-existing phosphorylated ends, followed by heat inactivation. Next, target regions were cleaved in vitro using Cas9 ribonucleoprotein complexes consisting of Alt-R S.p. HiFi Cas9 Nuclease V3 (Integrated DNA Technologies), Alt-R CRISPR-Cas9 tracRNA (Integrated DNA Technologies), and target-specific Alt-R CRISPR-Cas9 crRNAs (Integrated DNA Technologies). Following cleavage, Cas9 was heat inactivated and adapters were ligated to cleaved phosphorylated ends. Adapters from Ligation Sequencing Kit V14 SQK-LSK114 (Oxford Nanopore Technologies) were used in place of the adapter included in the Cas9 kit. Following adapter ligation, samples were cleaned up using AMPure XP Beads and loaded onto a R10.4.1 flow cell on a MinION Mk1C (Oxford Nanopore Technologies) for sequencing