GSM6263112: Fig4D CmYLCVp ribozyme A form PDS3gR10 WTCasphi rep1; Arabidopsis thaliana; OTHER
Amplicon sequencing: DNA was extracted from protoplast samples and leaves of transgenic plants with Qiagen DNeasy plant mini kit. The amplicon was obtained using two rounds of PCR. Amplification primers for the first round of PCR were designed to have the 3' sequence of the primers flanking a 200-300 bp fragment of the genomic area targeted by the guide RNA of interest. The 5' part of the primer contained a sequence which will be bound by common sequencing primers. After 25 cycles of the first round of PCR amplification, the reaction was purified using 1x Ampure XP beads (Beckman Coulter A63881). The eluate was used as template for the second round of PCR using the Phusion enzyme and 12 cycles of amplification. The second round of PCR was designed so that indexes were added to each sample. The samples were then purified using 0.8x Ampure XP beads. Part of the purified libraries were run on a 2% agarose gel to check for size and absence of primer dimer (fragments below 200 bp considered as primer dimer). Then amplicons were sent for sequencing with NovaSeq 6000 or iSeq 100 platform. Whole genome sequencing: DNA from single Arabidopsis seedlings was extracted with the Qiagen DNeasy plant mini kit and sheared to 300 bp size with a Covaris. Library preparation was performed with Tecan Ovation Ultralow V2 DNA-seq kit. The libraries were sequenced with the NovaSeq 6000 platform.