GSM8458391: IR D14; Mus musculus; RNA-Seq
The left and right kidneys were transferred to new sterile culture dishes and minced. 5 mL of sterile PBS and the dissociation solution from the Multi Tissue dissociation kit (Miltenyi, 130-110-203) were added to the dish. The tissue and liquid in the dish were transferred to EP tube and placed on a 37°C shaking incubator for digestion. After 30 minutes, the digested tissue was filtered with 40μm cell strainer. The filtered cell digestion solution was collected into 15 mL centrifuge tube. After centrifugation, the supernatant was discarded, cell pellet was incubated with 1 ml of RBC lysis buffer on ice for 3 mins. The reaction was deactivated by adding sterile PBS. After centrifugation, cell counting was performed, and PBS was added to dilute the cell concentration to 10^6 cells/mL to obtain a single-cell suspension. The cell count and viability were analyzed using the Countess AutoCounter (Invitrogen, C10227). The single-cell suspension, 10× barcoded gel beads, and oil were added to the wells of a single-cell sequencing chip according to the instructions, forming a droplet containing a single cell. Single-cell 5' sequence-related reagents were added to the system following a standardized process. Reverse transcription was then performed to generate cDNA, followed by PCR amplification for subsequent sequencing.