GSM7720823: kidney, MN, replicate 2, scRNAseq; Homo sapiens; RNA-Seq
For single cell isolation, renal samples were cut into 0.5mm2 pieces and then transferred to a 1.5mL-tube containing 1ml collagenase II and collagenase IV mixtures for digestion, subsequently filtered to remove cell debris and impurities. Next, 1 ml Red Blood Cell Lysis was added and incubated on ice for 2-10 min to lyse remaining red blood cells. After incubation, the cell suspension was centrifuged at 300g for 5 min at room temperature and then resuspended in 100 μl Dead Cell Removal MicroBeads. Subsequently, we removed dead cells using Dead Cell Removal Kit and resuspended cell samples in 1× PBS (0.04% BSA). Finally, cell suspension was centrifuged at 300 g for 3 min at 4 °C and resuspended in 50 μl of 1× PBS (0.04% BSA). The single-cell transcriptomic amplification and library preparation after single-cell suspension loaded onto the microfluidic chip were performed according to the manufacturer's instructions for 10×Genomics single-cell 3' kit (V3). The following mRNA trapping, reverse transcription, cDNA purification and amplification were prepared according to the manufacturer's protocol. The constructed library was sequenced using a Novaseq 6000 system.