Kapa hifi hot start plus dntps

All CENP-O coding sequences were cloned from testis or liver samples. The use of rat CENP-O to represent a divergent orthologue was motivated by the availability of a rat (Rattus norvegicus) tissue sample for cloning. Chimeric CENP-O constructs were designed based on their three-dimensional structure (AlphaFold database). Tissue was mechanically homogenized and total mRNA was isolated using TRIzol reagent (Invitrogen, 15596026), cDNA was prepared using reverse transcription (SuperScript III First-Strand Synthesis System; 18080051), amplified using construct-specific PCR primers (KAPA HiFi Hot Start plus dNTPs; Roche; KK2502), and inserted into the pGEMHE plasmid backbone (In-fusion kit; Takara; 638948). Each CENP-O construct was tagged with GFP at the C-terminus, separated by a linker of 5 glycines. Site-directed mutagenesis was performed using the Quik-Change Multisite Directed Mutagenesis kit (Agilent; 200515) to introduce point mutations. The identity of all constructs was confirmed using Sanger sequencing of the entire coding sequence, including the reporter gene.

All CENP-O coding sequences were cloned from testis or liver samples. The use of rat CENP-O to represent a divergent ortholog was motivated by the availability of a rat (Rattus norvegicus) tissue sample for cloning. Chimeric CENP-O constructs were designed based on their 3D structure (AlphaFold database). Tissue was mechanically homogenized and total mRNA was isolated using TRIzol reagent (15596026; Invitrogen); cDNA was prepared using reverse transcription (18080051; SuperScript III First-Strand Synthesis System), amplified using construct-specific PCR primers (KK2502; KAPA HiFi Hot Start plus dNTPs; Roche), and inserted into the pGEMHE plasmid backbone (638948; In-fusion kit; Takara). Primers were designed using SnapGene (Dotmatics) software and are listed in Table S6. Each CENP-O construct was tagged with GFP at the C-terminus, separated by a linker of five glycines. Site-directed mutagenesis was performed using the Quik-Change Multisite Directed Mutagenesis kit (200515; Agilent) to introduce R225G, C226T, T228A, N247G, and P261H mutations. Rat CENP-O with 11 mouse point mutations was synthesized (Genewiz). The identity of all constructs was confirmed using Sanger sequencing of the entire coding sequence, including the reporter gene.