title: GSM6804982: Renal single cell mRNA2; Homo sapiens; RNA-Seq
library_construction_protocol: Non-tumour portion were derived from partial or radical nephrectomies and collected into PBS on ice. Kidney samples were minced into 2-4mm and dissociated according to the manufacturer's protocol of Multi Tissue Dissociation Kit 1 (Miltenyi Biotech, Cat# 130-110-201). Red blood cells (RBC) were removed by incubating the sample with 2mL RBC lysis for 3 minutes on ice. The sample was enriched to live cells using Dead Cell Removal Kit (Miltenyi Biotech, Cat# 130-090-101), centrifuged at 300G for 7min, resuspended in MACS buffer (PBS containing 2% FBS and 5 mM EDTA pH 8.0) Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs 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 DynaBeads, 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.