Mighty cloning reagent set

Organoids were dissociated to single cells by trypsin treatment and single cells were seeded into 96‐well plates at 1 cell per well to obtain subclones. Total RNA was prepared from the subclones, and mRNA was reverse‐transcribed (RT) by SuperScript III (Thermo Fisher Scientific), amplified by polymerase chain reaction (PCR), and subcloned into the plasmid vector with Mighty Cloning Reagent Set (#6027; Takara Bio). PCR primer sequences are provided in Supplementary materials and methods. Subcloned cDNAs were sequenced using an Applied Biosystems 3500 genetic analyzer (Thermo Fisher Scientific).

Genomic DNA was extracted from calluses and regenerated plants with a DNeasy Plant Mini Kit (Qiagen, Germany) according to the supplier’s instructions. To detect genome editing in Gret1 within the VvMYBA1a promoter region, we conducted PCR using primers F1 and R1 to amplify the solo LTR, F1, and 5′-LTR-R to amplify the 5′-LTR, and 3′-LTR-F and R1 to amplify 3′-LTR (S1 Table). To confirm the PCR, primers PDS-MPF1 and PDS-MPR1, which amplify the endogenous PDS gene, were added to the PCR mixture. Solo LTR PCR products were extracted from the gel and cloned into a Mighty cloning Reagent set (Blunt end) (Takara, Japan) according to the supplier’s instructions, and cloned PCR products were transformed into Escherichia coli DH5α (Takara Clontech, Japan). Plasmids were isolated with a TempliPhi Rolling Circle Amplification Kit (GE Healthcare, UK), and sequenced with a BigDye X-Terminator 3.1 cycle sequencing kit (Applied Biosystems, USA) on an ABI3130x sequencer (Applied Biosystems, USA). We used primers F1 and R1 for sequencing solo LTR, 5′-LTR-R and 3′-LTR-F for the 5′-LTR, and 5′-LTR-F3IN and 3FIN-R for the 3′-LTR (S1 Table). To detect mutations in the Gret1-like sequence, we used primers Off3-F, Off3-R, 5′-LTR-F, and 5′-LTR-F3IN for PCR and direct sequencing (S1 Table).