Cloning and Validation of Truncated CENP-A

Genomic DNA from wild-type fission yeast cells was used as a template for the PCR amplification of N-terminally truncated CENP-A gene copies using Phusion polymerase (Thermo Fisher Scientific, Waltham, MA, USA), whereas plasmids pFA6a-6xGLY-3xFLAG-kanMX6 [49 (link)] and pFA6a-GFP(S65T)-kanMX6 [50 (link)] were used as templates [50 (link)] for FLAG and GFP protein tags, respectively. All PCR products were ethanol-precipitated and subjected to gel purification using a DNA gel extraction kit (Qiagen) according to manufacturer’s instructions. Both plasmid pREP41 and purified DNA (PCR products) were treated with restriction enzymes (New England Biolabs (NEB), Ipswich, MA, USA) and subjected to agarose gel purification, as described previously [28 (link)]. Digested gene inserts were ligated to pREP41 plasmids with T4 ligase (NEB), and transformed into TOP10 competent cells (Thermo-Fisher Scientific), before plating onto LB + Car agar plates (Luria–Bertani medium with 100 μg/mL carbenicillin (GoldBio, St. Louis, MO, USA). Positive transformants were then expanded by plasmid miniprep (Qiagen, Hilden, Germany) and extracted plasmids were sequenced using the Sanger method for confirmation of the correct gene sequences, frame, and orientation.