GSM8797487: Root cells, SCREW1; Arabidopsis thaliana; RNA-Seq
Roots of seven-day-old seedlings were cut approximately one centimeter from their tips, broadly diced with a scalpel blade, and then incubated in protoplast isolation solution (1.5% cellulase R10, 1.2% cellulase RS, 0.2% macerozyme R10, and 0.15% pectinase, in 600 mM mannitol, 2 mM MES hydrate, 10 mM KCl, 2 mM CaCl2, 2 mM MgCl2, and 0.1% BSA, pH 5.7) for 2.5 hr at 20°C on an orbital shaker set at 60 revolutions/min. The individual elicitor was applied 30 min before cutting the roots of the seedlings and was also added to the protoplast solution. Protoplasts were filtered through 40 μm cell strainers and centrifuged at 300 g for 3 mins. The supernatant was gently removed, and the pellet resuspended in 10 ml of 8% mannitol solution. This wash procedure was repeated again; the protoplasts were centrifuged again and resuspended in ∼500 μl of 8% mannitol solution. Single-cell RNA-seq libraries were prepared from fresh protoplasts according to the 10x Genomics Single Cell 3' Reagent Kit v3 protocol (10x Genomics, USA). For each sample, 10,000 cells were loaded in the 10x Genomics Chromium single-cell microfluidics device with the aim of capturing 7,000 cells. 11 cycles were used for cDNA amplification, as well as for final PCR amplification of the adapter-ligated libraries. Final library size and quality were checked on a DNA High Sensitivity Bioanalyzer chip (Agilent, USA), and libraries were quantified using the NEBNext Library Quantification Kit for Illumina. The scRNA-seq library sequencing was performed on the NextSeq platform (Illumina, USA).