GSM7898597: 3-week-old Th-MYCN+/-_biol rep 9; Mus musculus; RNA-Seq
Upon euthanization, the SMG of mice was meticulously dissected under the microscope. The SMG is anchored to the mesenteric artery and is proximal to the renal vein. Care was taken to detach the SMG using forceps without perturbing the adjacent blood vessels. Any attached fat was excised. The appearance of the dissected SMG was slightly white and transparen. This tissue was subsequently incubated in an equal ratio mix of collagenase IV (2.5 mg/mL, C5138, Sigma-Aldrich) and TrypLE Express Enzyme (12604013, Thermo Fisher Scientific) at 37°C for 40 min. Afterward, 10 volumes of D-PBS (045-29795, FUJIFILM Wako Chemicals) containing 1% BSA (15260037, Thermo Fisher Scientific), DNase I (0.1 mg/mL, DN25, Sigma-Aldrich), and 10 mM MgCl2 were added, and the tissue was dissociated using a fire-polished Pasteur pipette until no substantial particles remained visible. The resultant cell suspension was centrifuged to obtain a cell pellet, which was then reconstituted in 1% BSA/D-PBS for subsequent analyses. For scRNA-seq, libraries were prepared using Chromium Single Cell 3' Reagent Kits v1 (10x Genomics) according to the user guide. Due to the limited number of cells in an SMG, the target cell recovery was set at 1,200 cells per sample.