Protoplasts were transfected with 10 or 20 μg pCDB-Cas9-GFP-PDS or pKAR6 in at least three independent experiments. The pKAR6 vector (Robert Blanvillain, unpublished data) (Thomson et al., 2011 (link); Supplementary Table 2 ) expresses GFP under a 35S promotor and was used as a positive control (PC) for protoplast transfection. Protoplasts transfected without vector (Negative Control 1; NC1) and protoplasts without the addition of both PEG and vector (Negative Control 2; NC2), were used as negative controls. After transfection, 1 mL of 0.5 M mannitol was added to the protoplast pellet and the protoplast suspension was transferred into a 6-well plate and cultured in the dark at 23 ± 2°C on an orbital shaker (30 rpm, 20 h). Next, the protoplast suspension was transferred to an Eppendorf tube, centrifuged for 5 min at 80 g in a swing out centrifuge and supernatant was removed. Twenty μL of the protoplast suspension was transferred to a Bürker chamber and analyzed with a Zeiss AxioImager M2 fluorescence microscope equipped with an Axiocam MRm camera and ZEN software and magnification 200 × (Carl Zeiss MicroImaging, Belgium). Transfection efficiencies were calculated as the ratio of the number of GFP expressing protoplasts (GFP Zeiss filter set 10 (excitation 489 nm, emission 509 nm) to the total number of living protoplasts (based on round shape under bright field microscopy).
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