GSM6926991: RGSV 21dpi, scRNAseq; Oryza sativa; RNA-Seq
Rice seedlings tested to be infected with the virus were taken out, the stem bases were cut 1.5cm (7dpi), 2.0cm (14dpi), and 2.5cm (21dpi), then chopped and soaked in mannitol. The stems segments were enzymatically (1.5% Cellulose RS, 0.75% Macerozyme R-10, 10mM MES (pH 5.7 with KOH), 0.4M Mannitol) hydrolyzed for 4 hours. Next, the hydrolysate is filtered through a cellular filter with a diameter of 40 microns, the filtered liquid was centrifuged at 100g for 5min at 4℃, supernatant was removed, and the precipitation was cleaned twice with W5 solution(2mM MES pH5.7 with KOH, 154mM NaCl, 125mM CaCl2, 5mMKCl). The collected protoplasts were finally re-suspended with a 5%PBS buffer and adjusted to a concentration of 500 to 1000 cells/μL. The protoplasts are kept on ice for later use. The scRNA-Seq libraries were generated using the 10X Genomics Chromium Controller Instrument and Chromium Single Cell 3' V3 Reagent Kits (10X Genomics, Pleasanton, CA). Briefly, cells were concentrated to approximately 1000 cells/uL and loaded into each channel to generate single-cell Gel Bead-In-Emulsions (GEMs). After the RT step, GEMs were broken and barcoded-cDNA was purified and amplified. The amplified barcoded cDNA was fragmented, A-tailed, ligated with adaptors and index PCR amplified. The final libraries were quantified using the Qubit High Sensitivity DNA assay (Thermo Fisher Scientific) and the size distribution of the libraries were determined using a High Sensitivity DNA chip on a Bioanalyzer 2200 (Agilent). All libraries were sequenced by illumina sequencer (Illumina, San Diego, CA) on a 150 bp paired-end run.