title: GSM8701325: BALB/c mice, Vehicle, biol rep 5; Mus musculus; RNA-Seq
library_construction_protocol: Collected tumor tissue were stored in the tissue storage solution immediately after resection and transferred on ice. Samples were washed three times and cut into approximately 1–2 mm3 pieces before being collected in the D-PBS buffer (Gibco), and projected to enzymatical digestion with Tumor Dissociation Kit (for mouse, Miltenyi Biotec) for 15 min on a rotor (140rpm/min) at 37℃ according to the manufacturer's protocol. The dissociated cells then passed through a 30μm SmartStrainer (Miltenyi Biotec) in the DMEM medium (Gibico) with 10% FBS and at 4℃ centrifuged at 500 g for 5 min. After the removal of the supernatant, the pelleted cells were re-suspended in 1 mL of red blood cell lysis buffer (Miltenyi Biotec) and incubated on ice for 2 min for red blood cell lysis before terminating the reaction with DMEM medium (Gibico, with 10% FBS). After being centrifuged at 400 g for another 5 min, the cell pellets would be re-suspended in sorting buffer (D-PBS supplemented with 1% FBS). After quality inspection, a single-cell suspension with a cell viability of more than 80%, a clumping rate of less than 10%, a background debris of less than 50%, and a moderate cell concentration was obtained. Subsequently, cell barcode-labeled beads and cells were passed through the 10X Genomics platform, and the cells and enzymes were encapsulated with oil droplets to form gel bead inemulsion. Cells are lysed within each independent oil droplet, and the ploy-A of the intracellular mRNA is labeled with a cell barcode on the bead. The reverse transcription is performed to form cDNA. Finally, the oil phase was removed and the next-generation sequencing library was constructed.
identifiers.namespace: GEO
identifiers.id: GSM8701325_r1