title: GSM8800033: 5dpi induced galls on tomato roots-Gall; Solanum lycopersicum; Meloidogyne incognita; RNA-Seq
library_construction_protocol: 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.