GSM6395362: DKD02; Homo sapiens; RNA-Seq
Each fresh kidney sample was cut into millimeter-sized pieces and enzymatically digested in Dulbecco's modified Eagle's medium (Gibco, 11995065) containing 5% fetal bovine serum (HyClone), 0.2% collagenase type IV (#C5138; Sigma–Aldrich), and 0.002% DNase I (Applichem, A3778.0050) for 5 min at 37°C with slight shaking. Digested tissues were then filtered through a 40μm pore size nylon mesh and centrifuged at 300g for 5min. The pellets were resuspended by culture medium and erythrocytes were lysed using a red blood cell lysis solution (MACS, 130-094-183) at 4℃ for 10 min, and then the cell suspension was centrifuged at 300g for 5min. After washed once in culture medium, the pellets were centrifuged at 300g for 5min, and the supernatant was removed. The pellets were then resuspended by 100μl culture medium, and the cell concentration and viability were assessed using Luna cell counter. The sequencing was performed on Illumina Nova 6000 PE150. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, 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 (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane magnetic beads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.