Peqgold cycle pure kit

Inserts for cloning were produced by PCR on cDNA from urediospore RNA using nested PCR. First-round PCR was in 10-µl reactions with 1 µl 10× buffer, 1 µl 10 mM dNTP mix, 0.5 µl 0.1 µM f1 primers each (see Table S1), 1-µl cDNA, and 0.1 µl Long PCR Enzyme Mix (Thermo Fischer Scientific). Second round PCR was in 50-µl reactions with 5 µl 10× buffer, 5 µl 10 mM dNTP mix, 2.5 µl f2 primers each, 2 µl PCR product from the first round reaction, and 0.5 µl Long PCR Enzyme Mix. Cycling conditions were the same for both PCRs: initial denaturation 3 min 94°C, then 35 cycles with 20 s 95°C, 30 s 55°C, and 60 s 68°C; final elongation 7 min 68°C. Second round PCR products were purified using the peqGOLD Cycle-Pure Kit (PEQLAB Biotechnologie GmbH, Erlangen, Germany) and inserted into the pCR^™8/GW/TOPO^® plasmid using the pCR^™8/GW/TOPO TA Cloning^® Kit (Invitrogen, Life Sciences, Carlsbad, CA, USA), following the instructions of the manufacturer and using 4 µl of the purified PCR product. All constructs were verified by sequencing. For production of constructs without the signal peptide, the inserts were PCR amplified again using a third set of primers and again inserted into the pCR^™8/GW/TOPO^® plasmid. All three sets of primers are supplied in Table S1.

For next-generation sequencing analyses (NGS) of the ADH1 locus, the genomic DNA of a pool of 40 primary transformants was extracted. To analyze repair patterns and SaCas9 efficiency, primers with 6 bp overhangs (AS17-AS20, see Supplementary Table 2 for oligonucleotide sequences) were designed to amplify a fragment containing the target site. For NGS analysis of the 45S rDNA ITS locus, DNA extraction of only the floral tissue was performed. Primer with 32 and 33 bp overhangs was designed (AS29 and AS30) for amplification of the target site. The 300–500 bp amplicons were generated using Q5 High-Fidelity DNA polymerase (New England Biolabs) and later purified using the peqGOLD Cycle Pure Kit (Peqlab). The amplicons were sequenced with the Illumina HiSeq platform at GATC Biotech. The quality of the reads was verified using the CLC Genomics Workbench (Qiagen Bioinformatics) and later analyzed using the CRISPR RGEN TOOL (Cas analyzer^{39 (link)}) (http://www.rgenome.net/cas-analyzer/#!). Parameters were set for minimum frequency (n) = 1 and comparison range R = 200, no wild-type marker (r). Afterward, the data were analyzed using R Studio and Excel.