Amplicon sequencing analysis of MsPLC4 predicted on-target mutation site
Genomic DNA from leaf tissue was extracted using the BioSprint 96 DNA Plant Kit (Qiagen Inc., Toronto, ON). Initial PCR amplifications were performed using Platinum SuperFi Green PCR Master Mix, 0.2 M of each primer and approximately 5-200 ng template DNA from mutant and wild-type genotypes in a total reaction volume of 25 L. Thermal cycling conditions included an initial denaturation of 98C for 30s, 25 cycles of 98C for 10s, 60C for 10s, and 72C for 30s, and a final extension at 72C for 1 min. Subsequently, a second round of PCRs was carried out using 5 L of a 1:20 dilution of the initial PCR products, Platinum SuperFi Green PCR Master Mix, along with 0.2 M of indexed forward and reverse primers as described above. The resulting amplicons were purified using the AMPure XP bead-based cleanup method (Beckman Coulter Inc., Brea, CA) and paired-end sequencing (300 bp) was performed using Illumina MiSeq i100 technology by a service provider (Integrated Microbiome Resource, Dalhousie University, Halifax, NS).