Cas9 target sequencing of Mus musculus: Knock-In hAce2 gene
High molecular weight DNA was prepared for targeted sequencing using the Cas9 Sequencing Kit (SQK-CS9109, ONT, UK), with 5 g being dephosphorylated for specificity toward regions of interest (ROIs). RNP complexes targeted DNA near the ROIs for cleavage, followed by A-tailing, purification, and adapter attachment. Sequencing was conducted on R9.4.1 flow cells (ONT) using adaptive sampling to focus on 15 kb around the ROIs, based on the Mus musculus genome (mm10). Nanopore sequencing was performed on a MinION Mk1c, with or without adaptive sampling, for a duration of 72 hours, generating Fast5 and FASTQ files for analysis. Reads covering the ROIs were extracted and used for downstream analysis.