GSM7688874: Sperm_untreated_NanoNOME_Cas9_targeted; Mus musculus; OTHER
About 2 million sperm were on-ice lysed with hypotonic lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween-20, and 0.01% digitonin) for 10 min. Sperm were pelleted down and resuspended in 500 ul of 1x GpC buffer containing 150 μl of 1 M sucrose, 1.5 μl of 32 mM S-adenosylmethionine (SAM; NEB, B9003) and 50 μl of M.CviPI (NEB, no. M0227L). The suspension was carefully mixed and incubated for 7.5 min at 37 °C, followed by a boost with an additional 25 μl of M.CviPI and 1.5 μl of SAM for 7.5 min. The reaction was stopped by the addition of 25 μl of 10% SDS, 5 μl of 0.5 M EDTA, 6 μl of 20mg/mL proteinase K, and 28 μl of 1M DTT followed by overnight incubation at 55 °C. The total sperm DNA was recovered by PCI and ethanol precipitation. For whole-genome nanopore sequencing, 1 ug of sperm DNA was used for library preparation using Oxford Nanopore Ligation-based library prep kit (ONT, SQK-LSK110). For target-enriched sequencing, single-stranded CRISPR–Cas9 DNA oligonucleotides (20 nt) were designed using the ChopChop v.3 designer tool (chopchop.rc.fas.har- vard.edu) and purchased from IDT. sgRNA were prepared using EnGen@sgRNA synthesis kit (NEB, no. E3322S). RNP complexes were assembled individually by combining 8 μl of nuclease-free water, 1.5 μl of NEBuffer r3.1 (NEB), 3 μl of 300 nM sgRNA, 1 μl of Cas9 Nuclease (NEB, no. M0386), followed by a 20-min incubation at 25 °C. The different RNP complexes were then pooled in a single tube. Per experiment, 3-4 μg of sperm gDNA was dephosphorylated with 10 μl of rSAP (NEB, no. M0371S) and 16 μl of 10× rCutSmart buffer (NEB, no. B6004S) for 30 min at 37 °C, then heat inactivated at 65 °C for 5 min. DNA was then purified through PCI and ethanol precipitation. The dephosphorylated DNA sample was then combined with the pool of RNP complexes in a 1:9 ratio. The reaction was incubated for 30 min at 37 °C to enable Cas9 cleavage. After digestion, Cas9 was inactivated by the addition of a 1/25 volume of 20 mg/mL proteinase K followed by a 15-min incubation at 55 °C. Cleaved and purified DNA was purified by PCI and ethanol precipitation and was subjected to dA-Tailing using Klenow Fragment (NEB, no. E6053). Reactions were incubated for 30 min at 37 °C and immediately subjected to adapter ligation as per the manual of Oxford Nanopore Ligation-based library prep kit (ONT, SQK-LSK110).