GSM8594831: ath_ev_rep2; Arabidopsis thaliana; RNA-Seq
Rosette leaves were harvested, sliced with a razor, and incubated in enzyme solution (1 % cellulase R10, 0.2 % macerozyme R10 (Yakult Honsha), 0.4 M mannitol, 20 mM MES pH 5.7, 20 mM KCl, 10 mM CaCl2, and 0.1 % BSA) for 90 min. Cells were strained through a 70 µM filter, collected by centrifugation at 300 g, and washed with W5 buffer (150 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES pH 5.7, 5 mM glucose). Cells were collected by centrifugation and transferred to MMg solution (400 mM mannitol, 10 mM MgCl2, 4 mM MES pH 5.7) for transfection. Cells were diluted to a cell density of 6 x 105 cells/ml with MMg and mixed with 20 µl plasmid DNA (20-50 µg DNA) in a 2 mL microcentrifuge tube. 120 ul PEG solution (40 % PEG4000, 200 mM mannitol, 100 mM CaCl2) was added to the cells and mixed by inversion. After incubating for 2-5 min, W5 buffer was added, and the cells were washed twice in W5 buffer. Cells were then transferred to a 48-well plate and incubated overnight with shaking Illumina library preparation was performed using the NEBNext Ultra II RNA Library Prep Kit for Illumina (NEB #E7770L) with NEBNext Multiplex Oligos for Illumina (NEB #E6609S) according to the manufacturer's instructions.