Sqk lsk109

Isolated nuclei were subjected to GpC methylation using M.CviPI GpC methyltransferase (NEB) followed by proteinase K and RNaseA treatment. Genomic DNA was purified from the reaction using AMPure XP beads. A total of 6–12 µg of genomic DNA was dephosphorylated with rSAP (NEB), followed by AMPure XP bead purification. In vitro Cas9 incubation was performed to enrich the nanopore library for regions of interest using EnGen sgRNA synthesis kit (NEB) and Cas9 nuclease (NEB). Cleaved genomic DNA product was treated with NEBNext dA-tailing kit (NEB) and subjected to library preparation using nanopore adapter (ONT SQK-LSK109) and Quick T4 DNA ligase (NEB). Libraries were sequenced in MinION flow cell (ONT R9.4.1). Sequencing data were then mapped to a mouse genome in which castaneous SNPs were incorporated using minimap2 (2.24) (Li 2018 (link)). Variant tagging and haploid phasing of each sequencing read were performed using nanopolish (0.13.2) (Simpson et al. 2017 (link)) and whatshap (Patterson et al. 2015 (link)). Mapped data were split into each haplotype using bamtools (Barnett et al. 2011 (link)), and CpG methylation analysis was performed using nanopolish call methylation package. Data were visualized in IGV (Robinson et al. 2011 (link)).

Isolated nuclei were subjected to GpC methylation using M.CviPI GpC methyltransferase (NEB), followed by proteinase K and RNaseA treatment. Genomic DNA was purified from the reaction using AMPure XP beads. A total of 6–12 µg of genomic DNA was dephosphorylated with rSAP (NEB), followed by AMPure XP beads purification. In vitro Cas9 incubation was performed to enrich the nanopore library for regions of interest using EnGen sgRNA synthesis kit (NEB) and Cas9 nuclease (NEB). Cleaved genomic DNA product was treated with NEBNext dA-tailing kit (NEB) and subjected to library preparation using nanopore adapter (ONT SQK-LSK109) and Quick T4 DNA ligase (NEB). Libraries were sequenced in MinION flow cell (ONT R9.4.1). Sequencing data was then mapped to the castaneous SNPs incorporated mouse genome using minimap2 (2.24).^{69 (link)} Variant tagging and haploid phasing of each sequencing read were performed using nanopolish (0.13.2)^{70 (link)} and whatshap.^{71 (link)} Mapped data was split into each haplotype using bamtools,^{72 (link)} and CpG methylation analysis was performed using nanopolish call-methylation package. Data was visualized in IGV.^{73 (link)}