title: Cas9-targeted amplification-free ONT sequencing of Sickle Cell Disease CD34 cells : DBE editing with gRNA20 and gRNA53
design: High molecule weight DNA was extracted from HSPC patient cells with the Nanobind CBB kit (PacBio, catalog no. 102-301-900) according to manufacturers instructions. DNA was size selected using Short Read Eliminator XS kit (PacBio, catalog no. 102-208-200) and quantified using the Qubit fluorometer (Thermo Fisher Scientific). 5g of DNA were used for the library preparation using the Cas9 Sequencing Kit (Oxford Nanopore Technologies-ONT, SQK-CS9109) and following the Cas9-mediated PCR-free enrichment protocol (version: CAS_9106_v109_revC_16Sep2020) available through ONT. Libraries were loaded onto MinION flow cells with R9.4.1 nanopores (ONT, catalog no. FLO-MIN106D). One flow cell was used per biological condition and run on Minion using MinKNOW software for 72h.  . Basecalling was performed on pod5 files using Dorado 0.8.3 and model dna_r9.4.1_e8_hac@v3.3 to generate fastq files.