GSM8800031: 5dpi-gall-neighboring tomato root tissues-Control; Solanum lycopersicum; RNA-Seq
Frozen tissue samples from tomato root were transferred to SeqMatic LLC (Fremont, CA, USA) in dry ice for nuclei isolation, library preparation, and sequencing. Frozen tissue was carefully transferred to a gentleMACS M tube that was filled with 5 ml of Honda buffer (2.5% Ficoll 400, 5% Dextran T40, 0.4 M sucrose, 10 mM MgCl2, 1 µM DTT, 0.5% Triton X-100, 1 tablet/50 ml cOmplete Protease Inhibitor Cocktail (Roche), 0.4 U µl−1, Protector RNase Inhibitor (Sigma-Aldrich), 25 mM Tris-HCl, pH 7.4). This buffer composition enables efficient lysis of cell membranes while keeping the nuclear membranes intact. The M tubes were placed onto a gentleMACS Dissociator, where a specific program was executed at 4°C to disrupt the tissue and release the nuclei. The resulting suspension was filtered through a 70-µm strainer and centrifuged at 700 g for 10 min at 4°C. Finally, the pellet was resuspended carefully in 500 µl Honda buffer and filtered through a 30-µm strainer. scRNA-seq libraries were generated using the Chromium Next GEM Single Cell 3ʹ Kit v3.1 following the manufacturer protocol (Manual part CG000315, Rev E; 10x Genomics). Library quality was validated using an Agilent TapeStation 4200 (High Sensitivity D1000 ScreenTape to evaluate library composition and size). Preliminary spike-in sequencing was performed and samples were pooled to equal read counts. Final library concentration was confirmed using qPCR on a Roche LightCycler 96.