GSM8573309: WT Cis1,scRNAseq; Mus musculus; RNA-Seq
The collected kidneys were decapsulated, minced, and digested with 1 mg/ml type IV collagenase and DNase I for 40 minutes at 37°C with agitation. After digestion, samples were filtered through 70-µm cell strainers to remove cell aggregate. Then pelleted cells were suspended in red blood cell lysis buffer to lyse erythrocytes. Then cell pellets were suspended and filtered to obtain single cell suspensions for single-cell sequencing.The single-cell suspension was enriched using Dead Cell Removal Micro Beads. Library was performed according to the manufacturer's protocols(single cell 3' v2 protocol,10x Genomics).Briefy, kidney cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion.The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA.The pooled barcoded cDNA was amplified by PCR and the apropiated sized fragments were selected with SPRI select reagent for subsequent library construction. protocol,10x Genomics).