GSM9069565: Tm-22-ToB; Solanum lycopersicum; RNA-Seq
The leaf strips were subjected to vacuum infiltration for 5 minutes and then incubated in the dark at 27°C with gentle shaking (50 rpm) for 3 hours to facilitate digestion. The resulting suspension was filtered through a 40 μm cell strainer and centrifuged at 100 g for 5 minutes at 4°C to collect the protoplasts. After removing the supernatant enzyme solution, protoplasts were washed twice with W5 solution (154 mM NaCl, 125 mM CaCl2·2H2O, 5 mM glucose, 2 mM MES, 5 mM KCl, pH 5.7).Protoplasts were then resuspended in 0.8 M mannitol solution to minimize cell aggregation. Concentrations were determined using a microscope, and viability was assessed by staining with 0.04% (w/v) trypan blue. Following the manufacturer's protocol, protoplast suspensions were immediately loaded into the Chromium Controller Instrument (10× Genomics, Pleasanton, CA, USA) to generate single-cell Gel Bead-In-Emulsions (GEMs). Single-cell RNA sequencing (scRNA-seq) libraries were prepared from the resulting cDNA using the Chromium Next GEM Single Cell 3' Reagent Kits v3.1, following the vendor's user guide. Silane magnetic beads were employed to remove residual biochemical reagents and primers from the post-GEM reaction mixture. Full-length barcoded cDNAs were subsequently amplified by PCR to obtain sufficient material for library construction. The finalized libraries were adaptor-ligated for Illumina bridge amplification and quality-controlled using an Agilent 2100 Bioanalyzer. Sequencing was performed on an Illumina NovaSeq 6000 platform (Gene Denovo, Guangzhou, China) to generate paired-end 150 bp reads.