GSM8062588: dorsal root ganglia, CCI, rep2; Mus; RNA-Seq
Each sample was placed in a sterile Petri dish on ice. Fat, fibrous and necrotic areas were removed, and the tissue was subsequently minced to smaller pieces of less than 3 mm. Samples were first washed with phosphate-buffered saline (PBS), minced into small pieces (approximately 1mm3) on ice and enzymatically digested with collagenase Ⅱ (Worthington) for 60 min at 37°C, with agitation. Subsequently, the sample was filtered using a 70-mm nylon mesh (Miltenyi #130-095-823). 10 ml of this cell suspension was counted by Trypan Blue to determine the concentration of live cells. The sample was then centrifuged at 300g and 4℃ for 5 min, and the supernatant was discarded. The cell pellet was resuspended in 1 ml freezing media (Gibco) for long term cryopreservation in liquid nitrogen. Throughout the dissociation procedure, cells were maintained on ice whenever possible, and the entire procedure was completed in less than 1 hr. Cryopreserved single-cell suspensions were promptly thawed and prepared as outlined by the 10x Genomics Single Cell 3 v3.1 Reagent Kit user guide.