Hepta adapter

All procedures for generating somatic and germline transformants and crosses were done as described [106 (link), 107 (link)] with some changes. For the germline biolistic transformation, 100 μg of plasmid DNA was digested with restriction enzymes to release the targeting fragment, and used to coat gold particles (SeaShell S550d or Chempur 900040), which were then deposited onto seven microcarriers and fitted into the hepta adapter (Biorad). A biolistic shooting was performed 4 hours after mixing of CU428 and B2086 strains. The hepta adapter assembly was positioned on the third slot from the top inside PDS1000/He and the rapture disk had the value of 1800 psi. The conjugating cells were spread as a thin layer onto a 10-cm wide Petri dish containing a layer of Tris-Agar (10 mM Tris-HCl pH 7.4, 1.5% agar) and positioned at the bottom-most slot of PDS1000/He. To generate homokaryons, we either used the standard procedure based on two rounds of genomic exclusion or the short circuit genomic exclusion (SCGE) [115 (link)] with either B*VI or B*VII [83 (link)]. Specifically, the homokaryons for SUP1, CDKR1-KO, LF4A-KO_CDKR1-KO, ovGFP-LF4A and ovGFP-LF4A_CDKR1-KO were made using the SCGE. For genotyping the cdkr1^sup1 allele its sequence introduced an MboI restriction site that was detected in the 1 kb PCR product (with primers 5’-TGGTGATTTTGGATCAGCT-3’ and 5’-CTTGCTTTCCTCAAATAAAC-3’).