Col-0-EV-RFP.R2
Arabidopsis roots of 15-day-old seedlings were harvested and the cell wall was removed using cellulase and macerozyme (Yakult, Japan). For each TF construct or the empty vector control, 46 million cells were transfected and aliquoted into 3 replicate wells of a 24-well plate. After overnight incubation, transfected root protoplasts were treated with 5 mM KNO3. Next, to identify direct regulated targets, 35M cycloheximide was added 20min before a 10M dexamethasone treatment. Transfected cells were sorted by FACS into RFP-expressing populations 3h after dexamethasone treatment.Cells overexpressing the TF or empty vector cells were collected, RNA was extracted using the PureLink RNA Mini Kit according to the instructions of the manufacturer (Catalog #12183018A, Ambion)  and RNA-Seq libraries were prepared according to the manufacturer's instructions using the NEBNext Ultra RNA Library Prep Kit for Illumina.