GSM8262830: AKI day 28; Mus musculus; RNA-Seq
Each kidney sample from three mice was minced and digested using the MultiTissue dissociation kit (Miltenyi, 130-110-203), followed by homogenization throsyringe-based mechanical disruption. The kidney tissue was enzymatically digested using a mixture of collagenase I, collagenase IV, and hyaluronidase in 1640 me (Gibco, USA) at 37°C for 40 minutes while suppressing the response with 10% fetal bovine serum (FBS). The resulting dissociated solution was passed through 70-μm cell strainer and then was centrifuged at 400g for 5min at 4℃ to collect the cell pellet. To remove any remaining erythrocytes, the red blood cell (RBC) lysis solution (Miltenyi,130-094-183) was applied on ice. Finally, the single-cell suspension was obtained with over 90% viability as detected by Countstar. This process was performed by CapitalBio Technology in Beijing. The harvested cell suspension was processed with the Chromium single Cell G Chip Kit (10x Genomics). The reverse transcription was performed with the S1000TM Touch Thermal Cycler (Bio Rad) following a procedure of 53°C for 45 minutes, 85°C for 5 minutes and a subsequent hold at 4°. The official library kit (Single Cell 3' Library and Gel Bead Kit V3.1) was applied to construct the libraries intended for single-cell RNA-seq analysis.